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Spectral resolution in conjunction with polar plots improves the accuracy and reliability of FLIM measurements and estimates of FRET efficiency.
J Microsc. 2011; 244(1): 21-37.A spectrograph with continuous wavelength resolution has been integrated into a frequency-domain fluorescence lifetime-resolved imaging microscope (FLIM). The spectral information assists in the separation of multiple lifetime components, and helps resolve signal cross-talking that can interfere with an accurate analysis of multiple lifetime processes. This extends the number of different dyes that can be measured simultaneously in a FLIM measurement. Spectrally resolved FLIM (spectral-FLIM) also provides a means to measure more accurately the lifetime of a dim fluorescence component (as low as 2% of the total intensity) in the presence of another fluorescence component with a much higher intensity. A more reliable separation of the donor and acceptor fluorescence signals are possible for Förster resonance energy transfer (FRET) measurements; this allows more accurate determinations of both donor and acceptor lifetimes. By combining the polar plot analysis with spectral-FLIM data, the spectral dispersion of the acceptor signal can be used to ... [truncated at 150 words]
Matsubara S, Chen YC, Caliandro R, Govindjee, Clegg RM.
Photosystem II fluorescence lifetime imaging in avocado leaves: Contributions of the lutein-epoxide and violaxanthin cycles to fluorescence quenching.
J Photochem Photobiol B. 2011; 104(1-2): 271-284.Lifetime-resolved imaging measurements of chlorophyll a fluorescence were made on leaves of avocado plants to study whether rapidly reversible ΔpH-dependent (transthylakoid H(+) concentration gradient) thermal energy dissipation (qE) and slowly reversible ΔpH-independent fluorescence quenching (qI) are modulated by lutein-epoxide and violaxanthin cycles operating in parallel. Under normal conditions (without inhibitors), analysis of the chlorophyll a fluorescence lifetime data revealed two major lifetime pools (1.5 and 0.5ns) for photosystem II during the ΔpH build-up under illumination. Formation of the 0.5-ns pool upon illumination was correlated with dark-retention of antheraxanthin and photo-converted lutein in leaves. Interconversion between the 1.5- and 0.5-ns lifetime pools took place during the slow part of the chlorophyll a fluorescence transient: first from 1.5ns to 0.5ns in the P-to-S phase, then back from 0.5ns to 1.5ns in the S-to-M phase. When linear electron transport and the resulting ΔpH build-up were inhibited by treatment with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the major ... [truncated at 150 words]
Eichorst JP, Huang H, Clegg RM, Wang Y.
Phase differential enhancement of FLIM to distinguish FRET components of a biosensor for monitoring molecular activity of membrane Type 1 matrix metalloproteinase in live cells.
J Fluoresc. 2011; 21(4): 1763-1777. PMCID: PMC3637990Fluorescence lifetime-resolved imaging microscopy (FLIM) has been used to monitor the enzymatic activity of a proteolytic enzyme, Membrane Type 1 Matrix Metalloproteinase (MT1-MMP), with a recently developed FRET-based biosensor in vitro and in live HeLa and HT1080 cells. MT1-MMP is a collagenaise that is involved in the destruction of extra-cellular matrix (ECM) proteins, as well as in various cellular functions including migration. The increased expression of MT1-MMP has been positively correlated with the invasive potential of tumor cells. However, the precise spatiotemporal activation patterns of MT1-MMP in live cells are still not well-established. The activity of MT1-MMP was examined with our biosensor in live cells. Imaging of live cells was performed with full-field frequency-domain FLIM. Image analysis was carried out both with polar plots and phase differential enhancement. Phase differential enhancement, which is similar to phase suppression, is shown to facilitate the differentiation between different conformations of the MT1-MMP biosensor ... [truncated at 150 words]
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Bioassays, FLIM and FRET.
8th International Weber Symposium. Kauai, Hawaii. June 12-17, 2011.
This talk will discuss some bioassays are based on FRET and lifetime measurements (FLIM) for measuring intracellular oxidation reduction potentials and the enzymatic activity of a matrix metalloproteinase. Fluorescent protein hybrids have played a central role in the development of these bioassay probes. An overview with examples will also be given of some analysis techniques that can be coupled with FLIM in order to provide rapid and detailed multi-parameter analysis of cellular based measurements.
Kolossov VL, Spring BQ, Clegg RM, Henry JJ, Sokolowski A, Kenis PJA, Gaskins HR.
Development of a high-dynamic range, GFP-based FRET probe sensitive to oxidative microenvironments.
Exp Biol Med (Maywood). 2011; 236(6): 681-691. PMCID: PMC3158092We report the optimization of a novel redox-sensitive probe with enhanced dynamic range and an exceptionally well-positioned oxidative midpoint redox potential. The present work characterizes factors that contribute to the improved Förster resonance energy transfer (FRET) performance of this green fluorescent protein (GFP)-based redox sensor. The α-helical linker, which separates the FRET donor and acceptor, has been extended in the new probe and leads to a decreased FRET efficiency in the linker's reduced, 'FRET-off' state. Unexpectedly, the FRET efficiency is increased in the new linker's oxidized, 'FRET-on' state compared with the parent probe, in spite of the longer linker sequence. The combination of a lowered baseline 'FRET-off' and an increased 'FRET-on' signal significantly improves the dynamic range of the probe for a more robust discrimination of its reduced and oxidized linker states. Mutagenesis of the cysteine residues within the α-helix linker reveals the importance of the fourth, C-terminal cysteine and ... [truncated at 150 words]
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Fluorescence lifetime-resolved imaging.
Photosynth Res. 2009; 102(2-3): 143-155.This is a short account of fluorescence lifetime-resolved imaging, in order to acquaint readers who are not experts with the basic methods for measuring lifetime-resolved signals throughout an image. We present the early FLI (fluorescence lifetime imaging) history, review shortly the instrumentation and experimental design, discuss briefly the fundamentals of the measured fluorescence response, and introduce the basic measurement methodologies. We also emphasize the complex nature of the fluorescence response in FLI signals, and introduce certain analysis methods that are appropriate and informative for complex fluorescence decays. The advantages of model independent analyses are discussed and examples given.
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Fluorescence lifetimes: fundamentals and interpretations.
Photosynth Res. 2009; 101(2-3): 181-194.Fluorescence measurements have been an established mainstay of photosynthesis experiments for many decades. Because in the photosynthesis literature the basics of excited states and their fates are not usually described, we have presented here an easily understandable text for biology students in the style of a chapter in a text book. In this review we give an educational overview of fundamental physical principles of fluorescence, with emphasis on the temporal response of emission. Escape from the excited state of a molecule is a dynamic event, and the fluorescence emission is in direct kinetic competition with several other pathways of de-excitation. It is essentially through a kinetic competition between all the pathways of de-excitation that we gain information about the fluorescent sample on the molecular scale. A simple probability allegory is presented that illustrates the basic ideas that are important for understanding and interpreting most fluorescence experiments. We also briefly point ... [truncated at 150 words]
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Image analysis for denoising full-field frequency-domain fluorescence lifetime images.
J Microsc. 2009; 235(2): 221-237.Video-rate fluorescence lifetime-resolved imaging microscopy (FLIM) is a quantitative imaging technique for measuring dynamic processes in biological specimens. FLIM offers valuable information in addition to simple fluorescence intensity imaging; for instance, the fluorescence lifetime is sensitive to the microenvironment of the fluorophore allowing reliable differentiation between concentration differences and dynamic quenching. Homodyne FLIM is a full-field frequency-domain technique for imaging fluorescence lifetimes at every pixel of a fluorescence image simultaneously. If a single modulation frequency is used, video-rate image acquisition is possible. Homodyne FLIM uses a gain-modulated image intensified charge-coupled device (ICCD) detector, which unfortunately is a major contribution to the noise of the measurement. Here we introduce image analysis for denoising homodyne FLIM data. The denoising routine is fast, improves the extraction of the fluorescence lifetime value(s) and increases the sensitivity and fluorescence lifetime resolving power of the FLIM instrument. The spatial resolution (especially the high spatial frequencies not ... [truncated at 150 words]
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Fluorescence lifetime-resolved imaging: what, why, how - a prologue.
FLIM Microscopy in Biology and Medicine. By A Periasamy and RM Clegg (Editors). Chapman & Hall/CRC, pp. 3-34, 2009. ISBN: 9781420078909INTRODUCTION: Fluorescence lifetime-resolved imaging, FLI, acquires a fluorescence image whereby the dynamic response of the fluorescence decay is temporally resolved at every location (pixel) of the image. When specifically referring to measurements in a light microscope, the acronym is FLIM, where the "M" stands for microscopy. We will use the names interchangeably. FLI measurements are analogous to normal intensity fluorescence imaging measurements and are acquired on the same samples, except that information related to the fluorescence lifetime is recorded in addition to the normal measurement of the fluorescence intensity. One says that in FLI the fluorescence signal is "lifetime resolved" and "spatially resolved." The fluorescence lifetimes (or more often, the apparent fluorescence lifetime) can be determined with the temporal resolution of nanoseconds or less at every pixel of the recorded image. The spectroscopic lifetime-resolved information can be displayed at every pixel in image format. By considering the physical mechanisms that ... [truncated at 150 words]
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Frequency-domain FLIM.
FLIM Microscopy in Biology and Medicine. By A Periasamy and RM Clegg (Editors). Chapman & Hall/CRC, pp. 115-142, 2009. ISBN: 9781420078909INTRODUCTION: For frequency-domain FLIM (fluorescence lifetime imaging microscopy), the amplitude of the excitation light is modulated repetitively at high frequency (HF). Radio frequencies between 1 and 200 MHz are nominal and chosen in order for the fluorescence response (nanoseconds) to be sensitive to the frequency of repetition/modulation. The waveform of the HF modulation is often sinusoidal, but it can be any repetitive shape; such a repetitive waveform can be decomposed into multiple harmonics in a Fourier series, and each sinusoidal harmonic component is treated separately. Only the fundamental frequency is present if the excitation is a pure sinusoidal modulation; other waveforms (e.g., square waves or pulse trains from mode-locked lasers) contain multiple harmonics of the repetition frequency.
Chen YC, Spring BQ, Buranachai C, Tong B, Malachowski GC, Clegg RM.
General concerns of FLIM data representation and analysis: frequency-domain model-free analysis.
FLIM Microscopy in Biology and Medicine. By A Periasamy and RM Clegg (Editors). Chapman & Hall/CRC, pp. 291-339, 2009. ISBN: 9781420078909INTRODUCTION: In the first part of this chapter we develop several mathematical expressions describing fluorescence lifetime measurements, and show the close correspondence between the time and frequency domains (Lakowicz 1999; Valeur 2002, 2005). We then discuss how the underlying physical parameters are deciphered from the measured FLIM experiments and present some techniques of image analysis and display that are peculiar to FLIM.
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FLIM applications in the biomedical sciences.
FLIM Microscopy in Biology and Medicine. By A Periasamy and RM Clegg (Editors). Chapman & Hall/CRC, pp. 385-400, 2009. ISBN: 9781420078909INTRODUCTION: Fluorescence lifetime measurements acquired with a spectrofluorometer (nonimaging) or light microscope (imaging) are vital tools in biological and clinical sciences because of the valuable information contained in fluorescence lifetime. Fluorescence is inherently a dynamic phenomenon; the steady-state intensity averages the fluorescence signal over the decisive time when the fluorescence is dynamically interacting and communicating on a molecular scale with its immediate environment (usually less than 10 ns). All physical and chemical events that transpire within the lifetime of the excited state of the fluorophore, such as solvent relaxation, rotational freedom of the probe, Förster resonance energy transfer, other excited-state reactions, and dynamic quenching, affect the dynamic response of an electronically excited fluorophore.
Chen YC, Spring BQ, Buranachai C, Malachowski GC, Clegg RM.
What is behind all those lifetimes anyway, and where do we go from here?.
BiOS 2009. Part of SPIE Photonics West. San Jose, California, January 24-29, 2009.
Multiphoton Microscopy in the Biomedical Sciences IX (Proceedings of SPIE, Vol. 7183). By A Periasamy and PTC So (Editors). 7183-1, 2009. ISBN: 9780819474292Fluorescence lifetime-resolved imaging microscopy (FLIM) has made tremendous strides in the last two decades. Exciting applications are being presented weekly, an extensive diversity of instrumentation and commercial devices have appeared and have improved dramatically, sophisticated algorithms for analysis and interpretation are now available, and FLIM is being coupled to other imaging modalities, such as spectral dispersion and anisotropy. In other words, FLIM has matured considerably, and is approaching a point where it can be routinely applied by researchers who are not involved with the instrumentation and analysis side of things. The number of interested users in FLIM has almost certainly surpassed that of the audience that previously employed single channel fluorescence lifetime measurements (in a cuvette). The reason is, of course, the imaging capability of FLIM and its exciting possibilities for biological applications, especially FRET. In this lecture I will attempt to give an overview of where we have come ... [truncated at 150 words]
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Förster Resonance Energy Transfer - FRET what is it, why do it, and how it's done.
FRET and FLIM Techniques (Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 33). By TWJ Gadella (Editors). Elsevier Science, pp. 1-57, 2009. ISBN: 9780080549583The applications of Förster resonance energy transfer (FRET) have expanded tremendously in the last 25 years, and the technique has become a staple technique in many biological and biophysical fields. Many publications appear weekly using FRET and most of the applications use FRET as a spectroscopic research tool. In this chapter, we have examined some general salient features of resonance energy transfer by stressing the kinetic competition of the FRET pathway with all other pathways of de-excitation. This approach emphasizes many of the biotechnological and biophysical uses of FRET, as well as emphasizing the important competing processes and biological functions of FRET in photosynthesis.
Malachowski GC, Clegg RM, Redford GI.
Analytic solutions to modelling exponential and harmonic functions using Chebyshev polynomials: fitting frequency-domain lifetime images with photobleaching.
J Microsc. 2007; 228(3): 282-295.A novel approach is introduced for modelling linear dynamic systems composed of exponentials and harmonics. The method improves the speed of current numerical techniques up to 1000-fold for problems that have solutions of multiple exponentials plus harmonics and decaying components. Such signals are common in fluorescence microscopy experiments. Selective constraints of the parameters being fitted are allowed. This method, using discrete Chebyshev transforms, will correctly fit large volumes of data using a noniterative, single-pass routine that is fast enough to analyse images in real time. The method is applied to fluorescence lifetime imaging data in the frequency domain with varying degrees of photobleaching over the time of total data acquisition. The accuracy of the Chebyshev method is compared to a simple rapid discrete Fourier transform (equivalent to least-squares fitting) that does not take the photobleaching into account. The method can be extended to other linear systems composed of different functions. ... [truncated at 150 words]
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Fluorescence measurements of duplex DNA oligomers under conditions conducive for forming M-DNA (a metal-DNA complex).
J Phys Chem B. 2007; 111(33): 10040 -10052.M-DNA (a metal complex of DNA with millimolar concentrations of Zn2+, Co2+, or Ni2+ and basic pH) has been proposed to undergo electron transfer over long distances along the helix and has generated interest as a potential building block for nanoelectronics. We show that DNA aggregates form under solvent conditions favorable for M-DNA (millimolar zinc and pH = 8.6) by fluorescence correlation spectroscopy. We have performed steady-state Förster resonance energy transfer (FRET) experiments with DNA oligomers conjugated with 6-carboxyfluorescein and tetramethylrhodamine to the opposite ends of double-stranded DNA (dsDNA) molecules. Enhanced acceptor emission is observed for distances larger than expected for identical DNA molecules with no zinc. To avoid intermolecular FRET, the fluorescently labeled dsDNA is diluted with a 100-fold excess of unlabeled dsDNA. The intramolecular FRET efficiency increases 25-fold for a 30-mer doubly labeled duplex DNA molecule upon addition of millimolar concentrations of zinc ions. Without zinc, this oligomer ... [truncated at 150 words]
Ermolenko DN, Spiegel PC, Majumdar ZK, Hickerson RP, Clegg RM, Noller HF.
The antibiotic viomycin traps the ribosome in an intermediate state of translocation.
Nat Struct Mol Biol. 2007; 14: 493-497.During protein synthesis, transfer RNA and messenger RNA undergo coupled translocation through the ribosome's A, P and E sites, a process catalyzed by elongation factor EF-G. Viomycin blocks translocation on bacterial ribosomes and is believed to bind at the subunit interface. Using fluorescent resonance energy transfer and chemical footprinting, we show that viomycin traps the ribosome in an intermediate state of translocation. Changes in FRET efficiency show that viomycin causes relative movement of the two ribosomal subunits indistinguishable from that induced by binding of EF-G with GDPNP. Chemical probing experiments indicate that viomycin induces formation of a hybrid-state translocation intermediate. Thus, viomycin inhibits translation through a unique mechanism, locking ribosomes in the hybrid state; the EF-G-induced 'ratcheted' state observed by cryo-EM is identical to the hybrid state; and, since translation is viomycin sensitive, the hybrid state may be present in vivo.
Holub O, Seufferheld MJ, Gohlke C, Govindjee, Heiss GJ, Clegg RM.
Fluorescence lifetime imaging microscopy of chlamydomonas reinhardtii: non-photochemical quenching mutants and the effect of photosynthetic inhibitors on the slow chlorophyll fluorescence transient.
J Microsc. 2007; 226(2): 90-120.Fluorescence lifetime-resolved images of chlorophyll fluorescence were acquired at the maximum P-level and during the slower transient (up to 250 s, including P-S-M-T) in the green photosynthetic alga Chlamydomonas reinhardtii. At the P-level, wild type and the violaxanthin-accumulating mutant npq1 show similar fluorescence intensity and fluorescence lifetime-resolved images. The zeaxanthin-accumulating mutant npq2 displays reduced fluorescence intensity at the P-level (about 25–35% less) and corresponding lifetime-resolved frequency domain phase and modulation values compared to wild type/npq1. A two-component analysis of possible lifetime compositions shows that the reduction of the fluorescence intensity can be interpreted as an increase in the fraction of a short lifetime component. This supports the important photoprotection function of zeaxanthin in photosynthetic samples, and is consistent with the notion of a ‘dimmer switch’. Similar, but quantitatively different, behaviour was observed in the intensity and fluorescence lifetime-resolved imaging measurements for cells that were treated with the electron transport inhibitor ... [truncated at 150 words]
Ermolenko DN, Majumdar ZK, Hickerson RP, Spiegel PC, Clegg RM, Noller HF.
Observation of intersubunit movement of the ribosome in solution using FRET.
J Mol Biol. 2007; 370(3): 530-540.Protein synthesis is believed to be a dynamic process, involving structural rearrangements of the ribosome. Cryo-EM reconstructions of certain elongation factor G (EF-G)-containing complexes have led to the proposal that translocation of tRNA and mRNA through the ribosome, from the A to P to E sites, is accompanied by a rotational movement between the two ribosomal subunits. Here, we have used Forster resonance energy transfer (FRET) to monitor changes in the relative orientation of the ribosomal subunits in different complexes trapped at intermediate stages of translocation in solution. Binding of EF-G to the ribosome in the presence of the non-hydrolyzable GTP analogue GDPNP or GTP plus fusidic acid causes an increase in the efficiency of energy transfer between fluorophores introduced into proteins S11 in the 30 S subunit and L9 in the 50 S subunit, and a decrease in energy transfer between S6 and L9. Similar anti-correlated changes in energy ... [truncated at 150 words]
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The history of FRET: from conception through the labors of birth.
Reviews in Fluorescence (Vol. 2006). By JR Lakowicz and CD Geddes (Editors). Springer, New York, pp. 1-45, 2007. ISBN: 9780387293424Introduction: This chapter is an excursion into the historical development of energy transfer. This chapter is not concerned with a detailed review of applications, or a review of modem theoretical developments; this is available elsewhere (Van Der Meer et al, 1994; Wu and Brand, 1994; Clegg, 1996). The topic is the emergence of Förster resonance energy transfer FRET. I also examine the ideas, experiments and theories that formed the scientific backdrop that preceded and led up to FRET.
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A simple derivation of the luminescence anisotropy decay from randomly distributed cylinders rotating about a single axis.
J Fluoresc. 2006; 16(6): 761-771.A simple derivation is given of the expression describing the anisotropy decay of luminescence for a solution of molecules that can only undergo rotational diffusion about a single cylindrical axis. The usual derivations of the anisotropy decay for this cylindrical model have simply taken limiting cases of the equations resulting from the general treatment of the anisotropy decay of a completely anisotropic rotator or the rotation of an ellipsoid. The arguments presented here can be understood without the mathematical sophistication required to follow the general derivations for the rotational diffusion of a completely anisotropic rotator or ellipsoids. The underlying physical mechanisms leading to a multiple exponential decay of the fluorescence anisotropy signal from a single axis rotating cylinder are clearly shown by following this derivation. The resulting expression for the anisotropy decay is not new. However, the derivation is easily understood, and this article is meant as an introduction to ... [truncated at 150 words]
Chandler DE, Majumdar ZK, Heiss GJ, Clegg RM.
Ruby crystal for demonstrating time- and frequency-domain methods of fluorescence lifetime measurements.
J Fluoresc. 2006; 16(6): 793-807.We present experiments that are convenient and educational for measuring fluorescence lifetimes with both time- and frequency-domain methods. The sample is ruby crystal, which has a lifetime of about 3.5 milliseconds, and is easy to use as a class-room demonstration. The experiments and methods of data analysis are used in the lab section of a class on optical spectroscopy, where we go through the theory and applications of fluorescence. Because the fluorescence decay time of ruby is in the millisecond region, the instrumentation for this experiment can be constructed easily and inexpensively compared to the nanosecond-resolved instrumentation required for most fluorescent compounds, which have nanosecond fluorescence lifetimes. The methods are applicable to other luminescent compounds with decay constants from microseconds and longer, such as transition metal and lanthanide complexes and phosphorescent samples. The experiments, which clearly demonstrate the theory and methods of measuring temporally resolved fluorescence, are instructive and demonstrate ... [truncated at 150 words]
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Microsecond mixing with polarization and spectral resolution for fast reaction kinetics.
50th Annual Meeting of the Biophysical Society, Salt Lake City, Utah, 2006.
Biophys J. 2006; Suppl, 693-Pos/B559.An instrument capable of recording fluorescence spectra and fluorescence anisotropy images has been integrated with a micro-fabricated, turbulent flow mixer to resolve microsecond kinetics of biological reactions. The turbulent mixer is able to completely mix two pure reagents within several microseconds. The resulting fluorescence data, taken along the length of the exit channel, is an image of the time evolution of the fluorescence spectra (or fluorescence anisotropy) as the reaction progresses. Here we present some example reaction times that have been measured utilizing FRET and anisotropy. Diffusion limited reactions that have been used to quantify the dead time of the mixer are also presented.
Noomnarm U, Sutin JDB, Clegg RM.
Repetitive pressure-jump for relaxation kinetics.
50th Annual Meeting of the Biophysical Society, Salt Lake City, Utah, 2006.
Biophys J. 2006; Suppl, 692-Pos/B558.Relaxation kinetics follows the time course of a chemical system changing from an initial equilibrium state to a new equilibrium state as a result of an external thermodynamic perturbation. Because the perturbed chemical systems remain close to equilibrium, the analysis of even complex reaction systems can be analyzed by linear differential equations. In addition, thermodynamic information (eg DV and DH) is directly available. On the other hand, steady-state kinetics or pre-steady state kinetics start the reaction system far from equilibrium and often bypass important intermediates. Pressure changes of 1-50 bar usually cause a free energy perturbation of chemical systems several orders of magnitude smaller than result from temperature changes in the range of 1-5 K. Another great advantage of pressure, is that perturbations smaller than a few thousand bar, in contrast to temperature changes of even a few degrees, often change the aggregation state of a macromolecular complex without disrupting ... [truncated at 150 words]
Redford GI, Majumdar ZK, Sutin JDB, Clegg RM.
Properties of microfluidic turbulent mixing revealed by fluorescence lifetime imaging.
J Chem Phys. 2005; 123(22): 224504.We present a new method of measurement based on fluorescence lifetime imaging that reveals molecular-scale details of the mixing process in a continuous-flow turbulent microfluidic reactor. Our data provide a glimpse of the cascade to the minimal eddy size, followed by rapid diffusion involving the smallest eddies for final mixing.
Majumdar ZK, Sutin JDB, Clegg RM.
Microfabricated continuous-flow, turbulent, microsecond mixer.
Rev Sci Instrum. 2005; 76(12): 125103.We present a microfabricated, continuous-flow, turbulent mixing device that can mix two or more fluids to complete homogeneity on the molecular scale in the microsecond range. The current design is compact, portable, relatively simple to fabricate, adaptable for various measurement techniques, and consumes small sample volumes. The entire mixing process is observable and we use this feature to characterize the dependence of the progress of mixing on the flow velocity. We present details of the mixer's construction and optical data acquisition using fluorescence. Because the mixer is constructed using microfabrication technology, it is inexpensive and alterations are easy to explore. We show that the dependence of mixing times and pressure drop on the flow velocity agree well with theoretical expectations for turbulent pipe flow.
Hickerson RP, Majumdar ZK, Baucom A, Clegg RM, Noller HF.
Measurement of internal movements within the 30 S ribosomal subunit using Förster resonance energy transfer.
J Mol Biol. 2005; 354(2): 459-72.We have used Förster resonance energy transfer (FRET) to study specific conformational changes in the Escherichia coli 30 S ribosomal subunit that occur upon association with the 50 S subunit. By measuring energy transfer between 13 different pairs of fluorescent probes attached to specific positions on 30 S subunit proteins, we have monitored changes in distance between different locations within the 30 S subunit in its free and 50 S-bound states. The measured distance changes provide restraints for modeling the movement that occurs within the 30 S subunit upon formation of the 70 S ribosome in solution. Treating the head, body, and platform domains of the 30 S subunit as simple rigid bodies, the lowest-energy solution converges on a model that satisfies each of the individual FRET restraints. In this model, the 30 S subunit head tilts towards the 50 S subunit, similar to the movement found in comparing 30 ... [truncated at 150 words]
Eckhoff DA, Sutin JDB, Clegg RM, Gratton E, Rogozhina EV, Braun PV.
Optical characterization of ultrasmall Si nanoparticles prepared through electrochemical dispersion of bulk Si.
J Phys Chem B. 2005; 109(42): 19786-97.Studying the properties and stability of silicon nanoparticles (Si-np) in aqueous environments may lead to novel applications in biological systems. In this work, we use absorption and photoluminescence (PL) spectroscopy to characterize ultrasmall Si-np prepared through anodic etching and ultrasonic fractionation of a crystalline Si wafer. Their behavior is studied over time in 2-propanol and during treatments with water, NaOH, HCl, and H(2)O(2). The observed population is divided into two types of material: bright species consisting of well-etched Si-np, approximately 1 nm in diameter, and dark species derived from partially etched or aggregated Si structures. The dark material is seen by its scattering in the 2-propanol and water solutions and is largely removed via precipitation with the NaOH or HCl treatment. The bright material includes three distinct species with their respective emissions in the UV-B, UV-A, and hard-blue regions of the spectrum. The hard-blue PL is shown to have a ... [truncated at 150 words]
Majumdar ZK, Hickerson RP, Noller HF, Clegg RM.
Measurements of internal distance changes of the 30S ribosome using FRET with multiple donor-acceptor pairs: quantitative spectroscopic methods.
J Mol Biol. 2005; 351(5): 1123-45.We present analytical and experimental procedures for determining distance changes within the 30 S subunit of the Escherichia coli ribosome using Förster resonance energy transfer (FRET). We discuss ways to contend with complexities when using FRET to measure distance changes within large multi-subunit macromolecular complexes, such as the ribosome. Complications can arise due to non-stoichiometric labeling of donor and acceptor probes, as well as environmental effects that are specific to each conjugation site. We show how to account for changes in extinction coefficients, quenching, labeling stoichiometry and other variations in the spectroscopic properties of the dye to enable more accurate calculation of distances from FRET data. We also discuss approximations that concern the orientation of the transition moments of the two dye molecules, as well as the impact of other errors in the measurement of absolute distances. Thirteen dye-pair locations with different distances using 18 independent FRET pairs conjugated to ... [truncated at 150 words]
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Polar plot representation for frequency-domain analysis of fluorescence lifetimes.
J Fluoresc. 2005; 15(5): 805-15.We present applications of polar plots for analyzing fluorescence lifetime data acquired in the frequency domain. This graphical, analytical method is especially useful for rapid FLIM measurements. The usual method for sorting out and determining the underlying lifetime components from a complex fluorescence signal is to carry out the measurement at multiple frequencies. When it is not possible to measure at more than one frequency, such as rapid lifetime imaging, specific features of the polar plot analysis yield valuable information, and provide a diagnostic visualization of the participating fluorescent species underlying a complex lifetime distributions. Data are presented where this polar plot presentation is useful to derive valuable, unique information about the underlying component distributions. We also discuss artifacts of photolysis and how this method can also be applied to samples where each fluorescence species shows a continuous distribution of lifetimes. Polar plots of frequency-domain data are commonly used for ... [truncated at 150 words]
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Real-time fluorescence lifetime imaging and FRET using fast gated image intensifiers.
Molecular Imaging: FRET Microscopy and Spectroscopy (Methods in Physiology Series). By A Periasamy and RN Day (Editors). Academic Press, pp. 193-226, 2005. ISBN: 9780195177206Summary
This chapter discusses practical features and guidelines for Fluorescence lifetime imaging (FLI) and Förster resonance energy transfer (FRET) that maybe helpful in reading the literature and in interpreting FLI and FRET measurements of complex biological systems. Several reviews are already available (Clegg and Schneider, 1996; [Clegg, et al, 1996] and [Clegg, et al, 2003]; Periasamy et al., 1996; [So, et al, 1996] and [So, et al, 1998]; Schneider and Clegg, 1997; Gadella et al., 2001; Cubeddu et al., 2002; Hink et al., 2002; Peter and Ameer-Beg, 2004). However, we feel that the general reader and aspiring user of FLI with applications to FRET would benefit from a concise, coherent presentation of fundamental aspects of these measurements and interpretations of the data. We will not include particular results from a biological system, nor specifics of new instrumentation. Instead, we focus on basic descriptions of the photophysical measurements and the general characteristics ... [truncated at 150 words]
Clegg RM, Breusegem SY, Barry NP, Holub O, Govindjee, Redford GI.
How can one choose the best method for measuring FRET in a microscope with my biological system?.
Microscopy and Microanalysis 2005. Honolulu, Hawaii, USA, July 31-August 4, 2005.
Microsc Microanal. 2005; 11(Suppl 2).Which method is the best for imaging FRET? This is a common question. It is simply
answered if you have only one possibility for measuring FRET. However, even in this
case it is best to be aware of the pitfalls, as well as the advantages, for the different
methods. It can be difficult to quantify FRET in cells because of experimental artifacts
and complexities in determining the necessary photophysical parameters. There are many
reasons for these difficulties: photobleaching, distributions of distances, varying relative
angles between the donor and acceptor, aggregation of the labeled macromolecules, lack
of information about relative concentrations of the complementary FRET pairs in
different locations, stability of the image during data acquisition, less that 100% labeling
of the donor and acceptor, spatially varying index of refraction, pH and ionic strength,
different extents of static or dynamic quenching. Every effect contributes uniquely and to
a different extent to the various methods of FRET measurements. In this lecture we will
give ... [truncated at 150 words]
Majumdar ZK, Hickerson R, Noller HF, Clegg RM.
Measurements of internal distance changes of the 30s ribosome using FRET with multiple donor-acceptor pairs: quantitative spectroscopic methods.
6th International Weber Symposium. Kauai, Hawaii. July 22-28, 2005.
I will present analytical and experimental procedures for determining distance changes within the 30S subunit of the Escherichia coli ribosome using Förster resonance energy transfer (FRET). Ways to contend with complexities when using FRET to measure distance changes within large multi-subunit macromolecular complexes, such as the ribosome will be discussed. Complications can arise due to non-stoichiometric labeling of donor and acceptor probes, as well as environmental effects that are specific to each conjugation site. We show how to account for changes in extinction coefficients, quenching, labeling stoichiometry and other variations in the spectroscopic properties of the dye to enable more accurate calculation of distances from FRET data. Approximations that concern the orientation of the transition moments of the two dye molecules, as well as the impact of other errors in the measurement of absolute distances, will be discussed. Thirteen dye-pair locations with different distances using 18 independent FRET pairs conjugated ... [truncated at 150 words]
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Nuts and bolts of excitation energy migration and energy transfer.
Chlorophyll a Fluorescence: A Signature of Photosynthesis (Advances in Photosynthesis and Respiration, Vol. 19). By GC Papageorgiou and Govindjee (Editors). Springer, pp. 83-105, 2005. ISBN: 9781402032172The applications of Fluorescence Resonance Energy Transfer (FRET) have expanded tremendously in the last 25 years, and the technique has become a staple technique in many biological and biophysical fields. Our understanding of photosynthesis is tightly coupled to our understanding of the transfer of captured energy from the absorption of photons, and following the energy flow through the complex maize of chemical reactions utilizing this energy. Many of these steps involve resonance energy transfer. In this chapter, we have examined some general salient features of resonance energy transfer by stressing the kinetic competition of the FRET pathway with all other pathways of de-excitation. This approach emphasizes many of the biotechnological and biophysical uses of FRET, as well as emphasizing the important competing processes and biological functions of FRET in photosynthesis. Many publications appear weekly using FRET and most of the applications use FRET as a spectroscopic research tool. Photosynthesis holds ... [truncated at 150 words]
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Enhanced acceptor FRET facilitated by M-DNA.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 799-Pos/B638.FRET experiments have been carried out on duplex metal-complexed DNA molecules that are doubly labeled on the 3’-ends. Metal-complexed DNA, or M-DNA1 , has been reported to be formed in the presence of divalent cations Zn2+, Ni2+, and Co2+ associating with double-stranded DNA to form a DNA-metal complex. Increased efficiency of Förster acceptor-enhanced energy transfer over distances larger than expected for the corresponding length of the DNA molecules is observed. Different methods of measuring FRET are carried out, including FRET measurements normalized by the directly excited acceptor fluorescence, (ratio)A. This latter method avoids difficulties in interpretation that might obscure the interpretation of donor quenching. FCS and light scattering studies show the onset of co-precipitation of DNA-metal complexes and metal hydroxides. Careful controls are necessary to differentiate inter-strand FRET resulting from close proximity of labeled DNA in the aggregated state from intra-strand transfer within each individual doubly labeled DNA molecule. We ... [truncated at 150 words]
Buranachai C, Ha T, Clegg RM.
Single molecule study of dynamic characteristics of Tetramethyrhodamine-DNA.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 1705-Pos/B691.Tetramethylrhodamine exhibits multiple fluorescent isomeric binding states upon conjugation on double stranded DNA. This has been shown by several previous steady state and time-resolved fluorescence studies. We have used single molecule spectroscopy to study the dynamics of conformational changes of the dye that is attached to the end of double-stranded oligo-DNA molecules immobilized on a glass surface. Transitions between three different discrete levels of fluorescence intensity versus time were observed. The intensity distributions of the three defined states were well described by Gaussian functions. The rates of transition between fluorescence states were determined by fitting an exponential decay function to the dwell-time histogram giving transition rates ranging from 0.03 to 0.5 s-1. These unexpected long dwell times have not been observed by previous studies.
Majumdar ZK, Clegg RM, Noller HF, Hickerson RP.
Monitoring internal movements of the 30S E. Coli Ribosome with quantitative and kinetic FRET measurements.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 1981-Pos/B93.We have made steady state and kinetic measurements of 30S-50S association by monitoring changes in FRET between site-specifically labeled fluorescent probes. We have developed a quantitative protocol that enables reliable measurements of distance changes that can be used to visualize internal movements of ribosome structure. We show that relative distance changes can be accurately determined, by distinguishing random from systematic error in fluorescence measurements. We show experimentally that the error in distance, due to kappa-squared uncertainty, is smaller than predicted by simple cone angle models of a single dipole transition using only dynamic averaging. We are able to correlate kinetic FRET measurements of subunit association with modeled conformational changes that ensue upon formation of the 70S complex.
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Two-photon fluorescence imaging and reactive oxygen species detection within the epidermis.
Epidermal Cells: Methods and Protocols (Methods in Molecular Biology, Vol. 289). By K Turksen (Editors). Humana Press, pp. 413-421, 2005. ISBN: 9781592598304Two-photon fluorescence microscopy is used to detect ultraviolet-induced reactive oxygen species (ROS) in the epidermis and the dermis of ex vivo human skin and skin equivalents. Skin is incubated with the nonfluorescent ROS probe dihydrorhodamine, which reacts with ROS such as singlet oxygen and hydrogen peroxide to form fluorescent rhodamine-123. Unlike confocal microscopic methods, two-photon excitation provides depth penetration through the epidermis and dermis with little photodamage to the sample. This method also provides submicron spatial resolution such that subcellular areas that generate ROS can be detected. In addition, comparative studies can be made to determine the effect of applied agents (drugs, therapeutics) upon ROS levels at any layer or cellular region within the skin.
Rao S, Sutin JDB, Clegg RM, Gratton E, Nayfeh MH, Habbal S, Tsolakidis A, Martin RM.
Excited states of tetrahedral single-core Si29 nanoparticles.
Phys Rev B. 2004; 69(20): 205319.We dispersed bulk crystalline Si into identical hydrogenated nanoparticles with negligible impurities and defects, which provide the opportunity for detailed comparison between measurement and theory. The UV photoluminescence of a dispersion of 1 nm silicon particles was studied. Distinct bands appear in the emission spectra with the lowest peaks in wavelength identified to be at 400, 360, and 310 nm with optimal excitation at 3.7, 4.0, and 4.6 eV, respectively. The multiple photoluminescence bands are analyzed in terms of the molecularlike energy levels of one bulklike and two nonbulklike reconstruction configurations of the filled fullerene single-core Si29H24, calculated by quantum Monte Carlo calculations and by time-dependent density functional theory. The measured bands are in close agreement with the excited states of the ideal bulklike configuration. However, there is a possibility that some of the observed bands might originate from the nonbulklike reconstructions. The Stokes shifts are discussed in terms of ... [truncated at 150 words]
Hanson KM, Hayden PJ, Kubilus J, Clegg RM.
Detecting reactive oxygen species in skin using two-photon fluorescence imaging microscopy.
65th Annual Meeting of the Society for Investigative Dermatology. Providence, RI. April 28-May 1, 2004.
J Invest Dermatol. 2004; 122(3): A140, 839.Reactive oxygen species (ROS) contribute to skin photodamage including photoaging, immunomodulation, actinic keratosis and skin cancers. Because these highly-reactive derivatives of molecular oxygen are extremely short-lived and essentially non-emissive, they are difficult to detect directly. In addition, until recently with the realization of two-photon excited fluorescence (TPEF) imaging, the opaque and heterogeneous environment of the skin has inhibited detection and quantification of UV-induced ROS within the skin. We have developed a TPEF imaging method to detect the presence of ROS with 0.5 m spatial resolution and >100 m depth penetration. Dihydrorhodamine (DHR, 100 uL, 50 uM) is applied to the skin surface (Epiderm-200 (epidermis) and Epiderm-200FT (epidermis/dermis) (MatTek Corp.)) and incubated for 1 hr (37 oC, 5% CO2). DHR is non-fluorescent until it reacts with ROS and forms fluorescent rhodamine-123 (R123). Samples are imaged before and after UV irradiation (200-1600 J m2, 280-400 nm, solar simulator, Solar Light Co.). A ... [truncated at 150 words]
Buranachai C, Leuba S, Ordahl CP, Kun E, Clegg RM.
Histone H1 and DNA interaction study using several methods in fluorescence spectroscopy.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 3055-Pos/B307.Binding of histone H1 to DNA has several functions in addition to chromatin folding such as assisting the binding of PARP complex. Binding of histone H1 onto oligo-duplex DNA has been studied using FRET, fluorescence anisotropy, FCS and time resolve lifetime measurements. Quantitative FCS and anisotropy experiments of the histone-DNA interactions show multiple binding sites of DNA on each histone H1 molecule. The binding of DNA to histone H1 can be followed by analysis of the auto- and cross correlation curves. Quenching of fluorescence from dye attached to DNA is observed upon binding of histone H1.
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Measuring enhanced acceptor fluorescence as a probe for electron transfer in m-DNA.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 2443-Pos/B540.Some divalent cations, e.g. Zn2+, Ni2+, and Co2+, associate with duplex DNA at approximately millimolar concentrations to form a metal complex (M-DNA) that has been proposed to facilitate electron transfer over long distances.1 Quenching of a fluorescence donor was also observed over distance much greater than a typical Förster radius. We have carried out acceptor-enhanced energy transfer experiments, among other measurements, on double-stranded M-DNA that are doubly labeled on the 5’-ends in order to investigate this structure further. We observe increased emission of the fluorescent acceptor over distances larger than expected for Förster transfer. The energy transfer measurements are normalized by the (ratio)A method, unambiguously identifying the energy transfer. The normalized enhanced acceptor fluorescence, (ratio)A, avoids complications by alternate de-excitation pathways (other than Förster transfer) that would affect the donor in the excited state and obscure the interpretation of donor quenching alone. We present the analysis of these energy transfer ... [truncated at 150 words]
Majumdar ZK, Clegg RM, Noller HF, Hickerson RP.
Visualizing ribosomal movements with Förster resonance energy transfer.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 1644-Pos/B629.We have probed distances between various locations within the Escherichia Coli 30s ribosomal subunit as well as inter-subunit distances of the 70s ribosomal complex, in a number of different functional states, by measuring energy transfer between site-specifically labeled ribosomal proteins. The FRET distance information allows preliminary modeling for visualizing conformational changes of the 30s subunit upon association with the 50s subunit. Changes in energy transfer have also been observed between pre and post-translocational states of the 70s, complexed with mRNA, tRNAs and EF-G, which reveal internal movements of the ribosome that occur during translocation. The methods of measurement will be described, and results will be discussed in relation to crystal structures and cyro-electron microscopy. Supported by the NIH, PHS 5 P41-RRO3155, UIUC, and by UCSC.
Eckhoff DA, Sutin JDB, Rogozhina EV, Stuart JN, Sweedler JV, Braun PV, Clegg RM, Gratton E.
Effects of oxidation on the photoluminescence from silicon nanoparticles.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 3156-Pos/B409.Understanding the stability of silicon nanoparticles (Si-np) in oxidative environments is important to their use as a luminescent marker in biophysical applications. Treatments with NaOH, HCl, and UV radiation lead to substantial red-shifts in the emission from ultrasmall (~1.0 nm) Si-np. Modeling of the photoluminescence (PL) spectra shows the same set of three distinct, narrow (~0.47 eV), near-Gaussian emissions with their relative strengths varying among the treatments. From the expected chemical and structural effects of the treatments and the results of computational modeling, each distinct emission is correlated with its probable chemical origin. A simple model of surface oxidation captures the general behavior and PL properties of this system. These results offer the exciting possibility of tailoring the PL properties of Si-np through control of their surface chemistry. Supported by the NIH, PHS 5 P41-RRO3155, and by UIUC.
Hohng S, Wilson TJ, Tan E, Clegg RM, Lilley DMJ, Ha T.
Conformational flexibility of four-way junctions in RNA.
J Mol Biol. 2004; 336(1): 69-79.Helical junctions are common architectural features in RNA. They are particularly important in autonomously folding molecules, as exemplified by the hairpin ribozyme. We have used single-molecule fluorescence spectroscopy to study the dynamic properties of the perfect (4H) four-way helical junction derived from the hairpin ribozyme. In the presence of Mg(2+), the junction samples parallel and antiparallel conformations and both stacking conformers, with a bias towards one antiparallel stacking conformer. There is continual interconversion between the forms, such that there are several transitions per second under physiological conditions. Our data suggest that interconversion proceeds via an open intermediate with reduced cation binding in which coaxial stacking between helices is disrupted. The rate of interconversion becomes slower at higher Mg(2+) concentrations, yet the activation barrier decreases under these conditions, indicating that entropic effects are important. Transitions also occur in the presence of Na(+) only; however, the coaxial stacking appears incomplete under these ... [truncated at 150 words]
McKinney SA, Tan E, Wilson TJ, Nahas MK, Déclais AC, Clegg RM, Lilley DMJ, Ha T.
Single-molecule studies of DNA and RNA four-way junctions.
Nucleic Acids Chemistry and Biology: The 5th Cambridge Symposium, Queen's College, Cambridge, 31 August–3 September 2003.
Biochem Soc Trans. 2004; 32(1): 41-45.Branched helical junctions are common in nucleic acids. In DNA, the four-way junction (Holliday junction) is an essential intermediate in homologous recombination and is a highly dynamic structure, capable of stacking conformer transitions and branch migration. Our single-molecule fluorescence studies provide unique insight into the energy landscape of Holliday junctions by visualizing these processes directly. In the hairpin ribozyme, an RNA four-way junction is an important structural element that enhances active-site formation by several orders of magnitude. Our single-molecule studies suggest a plausible mechanism for how the junction achieves this remarkable feat; the structural dynamics of the four-way junction bring about frequent contacts between the loops that are needed to form the active site. The most definitive evidence for this is the observation of three-state folding in single-hairpin ribozymes, the intermediate state of which is populated due to the intrinsic properties of the junction.
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The vital contributions of Perrin and Förster.
Biophotonics Int. 2004; 11(9): 42-45.The theories of the pioneers of energy transfer, which provides useful insight into the mechanism and leads to a better appreciation of the broad applicability of the technique, are discussed. Förster resonance energy transfer (FRET) is used extensively to monitor macromolecule interactions, to determine molecular distances and to follow conformational changes. Perrin demonstrated that if the molecules were separated within the nonradiating near field of the donor dipole, energy could be transferred to the acceptor molecule. He assumed that identical molecules have exactly the same fundamental oscillation frequency, that is, the two Hertzian oscillators are in exact resonance.
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Bioconvertible vitamin antioxidants improve sunscreen photoprotection against UV-induced reactive oxygen species.
J Cosmet Sci. 2003; 54(5): 589-98.The ability of sunscreens and antioxidants to deactivate highly destructive reactive oxygen species in human skin has remained inconclusive. Two-photon fluorescence imaging microscopy was used to determine the effect of sunscreen/antioxidant combinations upon UV-induced ROS generation in ex vivo human skin. A sunscreen combination containing octylmethoxycinnamate (Parsol MCX) and avobenzone (Parsol 1789) at SPF 8 and SPF 15 was tested for its ability to prevent UV radiation from generating ROS in the viable epidermal strata of ex vivo human skin. A UV dose equivalent to two hours of North American solar UV was used to irradiate the skin. Each sunscreen reduced the amount of ROS induced in the viable strata by a value consistent with the SPF level. UV photons that were not absorbed/scattered by the sunscreen formulations generated ROS within the viable epidermal layers. The addition of the bioconvertible antioxidants vitamin E acetate and sodium ascorbyl phosphate (STAY-C 50) ... [truncated at 150 words]
Tan E, Wilson TJ, Nahas MK, Clegg RM, Lilley DMJ, Ha T.
A four-way junction accelerates hairpin ribozyme folding via a discrete intermediate.
Proc Natl Acad Sci USA. 2003; 100(16): 9308-13. PMCID: PMC170914The natural form of the hairpin ribozyme comprises two major structural elements: a four-way RNA junction and two internal loops carried by adjacent arms of the junction. The ribozyme folds into its active conformation by an intimate association between the loops, and the efficiency of this process is greatly enhanced by the presence of the junction. We have used single-molecule spectroscopy to show that the natural form fluctuates among three distinct states: the folded state and two additional, rapidly interconverting states (proximal and distal) that are inherited from the junction. The proximal state juxtaposes the two loop elements, thereby increasing the probability of their interaction and thus accelerating folding by nearly three orders of magnitude and allowing the ribozyme to fold rapidly in physiological conditions. Therefore, the hairpin ribozyme exploits the dynamics of the junction to facilitate the formation of the active site from its other elements. Dynamic interplay between ... [truncated at 150 words]
Behne MJ, Barry NP, Hanson KM, Aronchik I, Clegg RM, Gratton E, Feingold K, Holleran WM, Elias PM, Mauro TM.
Neonatal development of the stratum corneum pH gradient: localization and mechanisms leading to emergence of optimal barrier function.
J Invest Dermatol. 2003; 120(6): 998-1006.Although basal permeability barrier function is established at birth, the higher risk for infections, dermatitis, and percutaneous absorption of toxic agents may indicate incomplete permeability barrier maturation in the early neonatal period. Since stratum corneum (SC) acidification in adults is required for normal permeability barrier homeostasis, and lipid processing occurs via acidic pH dependent enzymes, we hypothesized that, in parallel with the less acidic surface pH, newborn SC would exhibit signs of incomplete barrier formation. Fluorescence lifetime imaging reveals that neonatal rat SC acidification first becomes evident by postnatal day 3, in extracellular "microdomains" at the SC- stratum granulosum (SG) interface, where pH-sensitive lipid processing is known to occur. This localized acidification correlated temporally with efficient processing of secreted lamellar body contents to mature extracellular lamellar bilayers. Since expression of the key acidifying mechanism NHE1 is maximal just prior to birth, and gradually declines over the first postnatal week, suboptimal ... [truncated at 150 words]
Redford GI, Gohlke C, Clegg RM.
Versatile rapid lifetime imaging.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2): 584, 2860-Pos/B459.A full-field fluorescence lifetime instrument using a homodyne frequency domain method will be presented. This instrument captures and displays real-time lifetime images at rates higher than 30 frames per second. It can be adapted to a microscope or an endoscope. Multiple frequencies are easily implemented. Immediate feedback and ease of use have been emphasized so that the device to can also be used for clinical diagnostic purposes. The instrument has been designed so that the benefits of lifetime imaging can be employed conveniently wherever standard fluorescence imaging is used. Data is shown for biological and biotechnological samples.
Majumdar ZK, Sutin JDB, Redford GI, Clegg RM.
Microsecond kinetics in a continuous flow turbulent mixer - detection with fluorescence intensity and fluorescence lifetime imaging.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 2321-Pos/B697.We have constructed a continuous-flow, turbulent mixing device that mixes two solutions to complete homogeneity in less than 12 microseconds. Novel microfabrication techniques have been employed for constructing the mixer in a glass substrate and these techniques will be discussed. The entire mixing process and kinetics can be observed in a steady state measurement with an optical microscope or other conventional optical system. We show how we use the mixing device together with lifetime and intensity imaging to monitor fast kinetic events of biological reactions.
Breusegem SY, Mackinnon AC, Barry NP, Lin X, Gratton E, Williams BD, Clegg RM.
In vivo visualization of protein-protein interactions in C. Elegans muscle attachment structures by FRET microscopy.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 105-Plat.Dense bodies and M-lines are highly ordered structures that attach actin, respectively myosin filaments in the body wall muscle cells of C. elegans to the basal lamina, hypodermis and cuticle. Using genetic techniques many proteins that localize at dense bodies and M-lines have been identified. To determine which proteins interact with each other in adult muscle attachments, we applied fluorescence resonance energy transfer (FRET) microscopy in living transgenic animals in which the proteins are fused pair-wise to the GFP-mutants CFP (cyan) and YFP (yellow). Occurrence of FRET between the CFP- and YFP-labels indicates a direct intermolecular interaction between the host proteins. FRET was measured by both steady-state and time-resolved two-photon excitation microscopy methods. We show that PAT-4, the C. elegans homolog of integrin-linked kinase, forms multimers and binds to PAT-6, a homolog of actopaxin, confirming results obtained from genetic and yeast two-hybrid studies. We discuss the advantages and disadvantages of ... [truncated at 150 words]
Tan E, Nahas MK, Wilson TJ, Clegg RM, Lilley DMJ, Ha T.
Dynamics of four-way junction enhances the active site formation of hairpin ribozyme.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 888-Pos/B140.The hairpin ribozyme catalyzes cleaving reaction when distant domains of the molecule interact and form the active site. The 4-way-junction structure of the native ribozyme enhances the active site formation 1000 fold compared to the minimal form, how this is achieved has been unclear. Using single molecule spectroscopy, we have discovered that the isolated form of this junction is highly dynamic displaying rapid switching behavior between different conformation. Comparison to the single molecule dynamics of hairpin ribozyme in various states of mutation indicate that the ribozyme inherits and exploits the conformer switching behavior of junction structure.
Clegg RM, Holub O, Gohlke C.
Fluorescence lifetime-resolved imaging: measuring lifetimes in an image.
Biophotonics, Part A (Methods in Enzymology, Vol. 360). By G Marriott and I Parker (Editors). Academic Press, pp. 509-42, 2003. ISBN: 9780121822637We have given an overview of what one can gain by lifetime-resolved imaging and reviewed the major issues concerning lifetime-resolved measurements and FLI instrumentation. Instead of giving diverse selected examples, we have discussed the underlying basic pathways of deexcitation available to the molecules in the excited state. It is by traversing these pathways that compete kinetically with the fluorescence pathway of deactivation–and therefore affect the measured fluorescence lifetime–that we gain the information that lifetime-resolved fluorescence provides. It is hoped that being aware of the diversity, of pathways available to an excited fluorophore will facilitate potential users to recognize the value of FLI measurements and inspire innovative experiments using lifetime-resolved imaging. FLI gives us the ability within a fluorescence image of measuring and quantifying dynamic events taking place in the immediate surroundings of fluorophores as well as locating the fluorescent components within the image. Just as measurements in cuvettes, lifetime-resolved imaging ... [truncated at 150 words]
Behne MJ, Meyer JW, Hanson KM, Barry NP, Murata S, Crumrine D, Clegg RM, Gratton E, Holleran WM, Elias PM, Mauro TM.
NHE1 regulates the stratum corneum permeability barrier homeostasis. Microenvironment acidification assessed with fluorescence lifetime imaging.
J Biol Chem. 2002; 277(49): 47399-406.The outermost epidermal layer, the stratum corneum (SC), exhibits an acidic surface pH, whereas the pH at its base approaches neutrality. NHE1 is the only Na(+)/H(+) antiporter isoform in keratinocytes and epidermis, and has been shown to regulate intracellular pH. We now demonstrate a novel function for NHE1, as we find that it also controls acidification of extracellular "microdomains" in the SC that are essential for activation of pH-sensitive enzymes and the formation of the epidermal permeability barrier. NHE1 expression in epidermis is most pronounced in granular cell layers, and although the surface pH of NHE1 knockout mice is only slightly more alkaline than normal using conventional pH measurements, a more sensitive method, fluorescence lifetime imaging, demonstrates that the acidic intercellular domains at the surface and of the lower SC disappear in NHE1 -/- animals. Fluorescence lifetime imaging studies also reveal that SC acidification does not occur through a uniform ... [truncated at 150 words]
Hanson KM, Behne MJ, Barry NP, Mauro TM, Gratton E, Clegg RM.
Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient.
Biophys J. 2002; 83(3): 1682-90. PMCID: PMC1302264Two-photon fluorescence lifetime imaging is used to identify microdomains (1-25 microm) of two distinct pH values within the uppermost layer of the epidermis (stratum corneum). The fluorophore used is 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), whose lifetime tau (pH 4.5, tau = 2.75 ns; pH 8.5, tau = 3.90 ns) is pH dependent over the pH range of the stratum corneum (pH 4.5 to pH 7.2). Hairless mice (SKH1-hrBR) are used as a model for human skin. Images (< or =50 microm x 50 microm) are acquired every 1.7 microm from the stratum corneum surface to the first viable layer (stratum granulosum). Acidic microdomains (average pH 6.0) of variable size (~1 microm in diameter with variable length) are detected within the extracellular matrix of the stratum corneum, whereas the intracellular space of the corneocytes in mid-stratum corneum (25 microm diameter) approaches neutrality (average pH 7.0). The surface is acidic. The average pH of the ... [truncated at 150 words]
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Observation and quantification of ultraviolet-induced reactive oxygen species in ex vivo human skin.
Photochem Photobiol. 2002; 76(1): 57-63.Two-photon fluorescence imaging is used to detect UV-induced reactive oxygen species (ROS) in ex vivo human skin in this study. ROS (potentially H202, singlet oxygen or peroxynitrite [or all]) are detected after reaction with nonfluorescent dihydrorhodamine-123 (DHR) and the consequent formation of fluorescent rhodamine-123 (R123). The cellular regions at each epidermal stratum that generate ROS are identified. R-123 fluorescence is detected predominately in the lipid matrix of the stratum corneum. In contrast, the strongest R123 fluorescence signal is detected in the intracellular cytoplasm of the viable epidermal keratinocytes. A simple bimolecular one-step kinetic model is used for estimating the upper bound of the number of ROS that are generated in the skin and that react with DHR. After ultraviolet-B radiation (280-320 nm) (UVB) equivalent to 2 h of noonday summer North American solar exposure (1600 J m(-2) UVB), the model finds that 14.70 × 10(-3) mol of ROS that react ... [truncated at 150 words]
Breusegem SY, Barry NP, Lin X, Gratton E, Clegg RM, Williams BD.
Protein interactions in C. Elegans muscle attachment structures studied in vivo by fluorescence resonance energy transfer (FRET).
Midwest Worm Meeting. June 28-30, 2002. Washington University in St. Louis.
Dense bodies and M-lines are highly ordered, multiprotein attachment structures that span the plasma membrane and function to link the myofilaments found in body-wall muscle cells to the underlying ECM. Assembly of these structures is essential for the viability of the organism, and recent work in a number of labs has identified several new dense body and M-line components that are essential for this process. A crucial step towards understanding the assembly process is the characterization of the many protein-protein interactions that occur within attachment structures. Biochemical analysis identified protein-protein interactions between PAT-4 and UNC-112, PAT-4 and PAT-6, and PAT-4 and UNC-97 in vitro, and currently we are attempting to measure and characterize these interactions in vivo. Toward this goal, we created transgenic animals in which different dense body and M-line attachment proteins are fused to either CFP (cyan) or YFP (yellow) and are analyzing Fluorescence Resonance Energy Transfer (FRET) ... [truncated at 150 words]
Hanson KM, Holub O, Gohlke C, Barry NP, Behne MJ, Gratton E, Clegg RM.
Imaging in skin and plants: using photons and fluorescence lifetimes to find the molecules and quantify the information.
8th Biophysical Society Annual Meeting and Frontier Biophysics Symposium, Taiwan, ROC, 2002.
Due to sophisticated technological advances and innovative applications of new technology, numerous optical imaging techniques are being developed that are highly informative, quantitative, flexible and which can be applied directly to biological and biophysical studies and to medical diagnostics. Fluorescence imaging is one of the fastest growing areas. Many measurements that were previously limited to cuvette type studies are now being incorporated into fluorescence microscopes and into medical diagnostics (such as the endoscope). This lecture will describe two techniques 1) “Fluorescence Lifetime-resolved Imaging” (FLI - where every pixel of the image contains the lifetime-resolved information) and its application to several biological areas, and 2) two-photon scanning imaging applied to skin diagnostic measurements. A brief general description of the various methods in our laboratory will be presented. The reason for making fluorescence lifetime measurements in images will be discussed. The necessity for real-time imaging is stressed, and the performance characteristics of ... [truncated at 150 words]
Behne MJ, Barry NP, Hanson KM, Meyer JW, Crumrine D, Clegg RM, Gratton E, Holleran WM, Elias PM, Mauro TM.
NHE1 regulates stratum corneum acidification and permeability barrier homeostasis: identification of acidic microenvironments with FLIM.
Biomedical Topical Meeting, Miami Beach, Florida, 2002.
Detecting impeded blood flow and locating the clot causing it is a major challenge in neurosurgery. We propose an instrument that uses near-infrared spectroscopy to simultaneously detect clots and measure blood flow.
Barry NP, Hanson KM, Gratton E, Clegg RM, Behne MJ, Mauro TM.
Applications of ultrafast lasers to two-photon fluorescence and lifetime imaging.
Commercial and Biomedical Applications of Ultrafast and Free-Electron Lasers (Proceedings of SPIE, Vol. 4633). By GS Edwards, J Neev, A Ostendorf, and JC Sutherland (Editors). pp. 50-61, 2002. Fluorescent probes have found widespread use in biomedical sciences. Particularly since they can be targeted to cellular compartments and further more can report on the properties of their environment such as calcium concentration. Near infrared ultrafast lasers find increasing use for fluorescence applications since femtosecond pulses with a few milliwatts of average power are sufficient to induce significant two photon fluorescence from the probe when focused into typical samples. The nonlinear optical excitation process allows sectioned imaging of 3-D samples without use of a confocal pinhole. In this paper we describe two aspects of multiphoton microscopy: the two- photon excitation cross section and the fluorescence lifetime. Of interest is the wavelength characterization of two-photon excitation cross-sections of fluorescence probes. We slowly modulate (~500Hz) the intensity envelope of the input laser pulse train and analyze the emission signal in terms of the amplitude and phase of the harmonics of this modulation. ... [truncated at 150 words]
Breusegem SY, Barry NP, Mackinnon AC, Lin X, Williams BD, Gratton E, Clegg RM.
Protein interactions in C. elegans muscle attachment structures studied in vivo by fluorescence resonance energy transfer (FRET).
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 491e.We are interested in deciphering the protein interactions in the focal adhesion-like structures that attach the muscles of C. elegans to its hypodermis and cuticle. To this goal we have created transgenic animals in which the proteins are fused pair-wise to the GFPmutants CFP (cyan) and YFP (yellow). Fluorescence resonance energy transfer (FRET) between the CFP- and YFP-labels will only occur if a direct intermolecular interaction between the host proteins exists. FRET measurements are done in vivo using several microscopy techniques. Using two-photon excitation we applied lifetime imaging, ratioimaging, donor photobleaching kinetics and recovery of the donor intensity after acceptor photobleaching. Using one-photon excitation we applied the intensity-based three-filter set method. To evaluate the accuracy of our results we performed control measurements on a Ca2+-sensitive cameleon construct purified in solution and expressed in bacteria, and on a CFP-YFP fusion construct expressed in C. elegans. The advantages and disadvantages of the ... [truncated at 150 words]
Hanson KM, Barry NP, Behne MJ, Mauro TM, Gratton E, Clegg RM.
Two-photon fluorescence lifetime imaging of the skin's stratum corneum pH gradient.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 494c.Two-photon fluorescence lifetime imaging is used to measure pH within the uppermost layer (stratum corneum) of hairless mice (SKH1-hrBR) skin. Lifetime measurements are made using the fluorophore BCECF whose fluorescence lifetime (τf) is pH sensitive. Following two-photon excitation at 820 nm, τf of BCECF is 2.72 ns at pH 4.5 and 3.93 ns at pH 8.5. Images (100 mm × 100 mm) were acquired every 1.7 mm from the stratum corneum surface through the viable lower strata. Acidic microdomains (average pH 5.9) of variable size (5 mm to 25 mm in diameter) are detected within the extracellular matrix of the stratum corneum, whereas the intracellular space approaches neutrality (average pH 6.7). The number of extracellular acidic microdomains decreases with each increasing stratum corneum depth reaching a minimum at the first viable epidermal layer (stratum granulosum). The average pH increases with depth due to the decrease in the ratio of acidic ... [truncated at 150 words]
vandeVen MJ, Holub O, Gohlke C, Govindjee, Valcke R, Ameloot M, Clegg RM.
Fast macroscopic chlorophyll fluorescence lifetime imaging of apple fruit skin.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 502a.Early detection of fruit skin storage diseases like bitterpit and storage scald is aided by macroscopic imaging of healthy and diseased tissue. Fast, whole area detection is necessary for quickly assessing fruit quality and for predicting storage potential. Macroscopic fluorescence lifetime imaging enhances contrast w.r.t. steady-state images and reduces geometrical effects. By monitoring photosystem II Chlorophyll a fluorescence it provides quick physiological information over an area not practically accessible by confocal (TPE) microscopy (Biophys. J., 80, 159a). Here we report using a fast-acquisition homodyning phase and modulation imaging fluorimeter (Biophys. J. 80, 169a, 428a) on a 1.8 cm diameter fruit skin area. Red and green/yellow sides of Malus Domesticus Borkh. x Golden Delicious, Jonagold and Granny Smith with and without surface defects were examined with a 690DF40 emission filter. A 6 μm single-mode fiber-optic directed the AOM modulated 488 nm laser light. Operating frequency was 80.652 MHz and total power ... [truncated at 150 words]
Behne MJ, Hanson KM, Barry NP, Clegg RM, Gratton E, Holleran WM, Elias PM, Mauro TM.
Correlation of extracellular enzyme activity and pH in Stratum Corneum by fluorescence lifetime imaging.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 501a.Acidification of the stratum corneum (SC) is essential for formation and maintenance of the mammalian epidermal permeability barrier, through its activation of SC pHdependent enzymes. Glucocerebrosidase (ß-GlcCer’ase) is active extracellularly in the lower SC. Its deletion results in abnormal SC structure and function both in human disease (Gaucher’s), and in transgenic models. Since ß-GlcCer’ase lipid processing requires an acidic milieu, its in situ activity reflects local pH. Paradoxically, the conventional view of the SC-pH gradient, increasing acidity from lower to outer layers, is contrary to the observed ß-
GlcCer’ase activity. Here, we report a positive correlation of pH and enzyme activity, using a Resorufin conjugate as a synthetic substrate for ß-GlcCer’ase, and simultaneous SC-pH measurement using two photon Fluorescence Lifetime Imaging (FLIM), and the pH sensitive dye BCECF. In contrast to flat electrode measurements, which are invasive, and cannot distinguish pHe vs. phi, we find acidic microdomains in lower layers of ... [truncated at 150 words]
Majumdar ZK, Sutin JDB, Clegg RM.
A continuous flow, turbulent mixer for studying fast reaction kinetics.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 483d.We have employed recently developed techniques for microelectromechanical systems (MEMS) for the construction of an ultra-fast, continuous-flow, turbulent mixer. The mixer constitutes a microfluidic system with channel dimensions of 20-80 micrometers made from PDMS (Polydimethylsiloxane elastomer). The design is tailored for making optical measurements of kinetics and the small scale is suitable for microscope or stereomicroscope observation, with the potential to consume volumes smaller than 100 microliters. This system is ideal for studying fast kinetics of biological macromolecules and chemical reactants initiated by a change in the chemical environment.
Breusegem SY, Clegg RM, Loontiens FG.
Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)4 DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex.
J Mol Biol. 2002; 315(5): 1049-61.The binding of Hoechst 33258 and DAPI to five different (A/T)4 sequences in a stable DNA hairpin was studied exploiting the substantial increase in dye fluorescence upon binding. The two dyes have comparable affinities for the AATT site (e.g. association constant K(a)=5.5 x 10(8) M(-1) for DAPI), and their affinities decrease in the series AATT >> TAAT approximately equal to ATAT > TATA approximately equal to TTAA. The extreme values of K(a) differ by a factor of 200 for Hoechst 33258 but only 30 for DAPI. The binding kinetics of Hoechst 33258 were measured by stopped-flow under pseudo-first order conditions with an (A/T)4 site in excess. The lower-resolution experiments can be well represented by single exponential processes, corresponding to a single-step binding mechanism. The calculated association-rate parameters for the five (A/T)4 sites are similar (2.46 x 10(8) M(-1) s(-1) to 0.86 x 10(8) M(-1) s(-1)) and nearly diffusion-controlled, while the ... [truncated at 150 words]
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FRET tells us about proximities, distances, orientations and dynamic properties.
J Biotechnol. 2002; 82(3): 177-9.Excerpt: A major challenge in biological science is to determine the spatial juxtapositions and distributions of molecular and supramolecular components that constitute biological structures. A great deal, if not the major part, of biology happens at the interface between interacting molecules. Techniques that inform us about these molecular interactions are of crucial importance...
Breusegem SY, Sadat-Ebrahimi SE, Douglas KT, Bichenkova EV, Clegg RM, Loontiens FG.
Experimental precedent for the need to involve the primary hydration layer of DNA in lead drug design.
J Med Chem. 2001; 44(16): 2503-6.The increase in fluorescence on binding ofm-phenyl substituted hydroxy derivatives of Hoechst 33258 with poly-[d(A-T)], d(CGCGAATTCGCG)2, and with the corresponding T4-looped 28-mer AATT hairpin was used to monitor binding by equilibrium titrations and stopped-flow kinetics. Replacing the p-OH substituent of Hoechst 33258 (association constant Ka ) 5.2 × 10^8 M-1 for 28-mer hairpin) by m-OH increases the AATT site binding energy by 1.1 kcal mol-1, Ka ) 3.8 × 10^9 M-1. Addition of a second m-hydroxy group (bis-m-OH Hoechst) further strengthens binding, giving Ka ) 1.9 × 10^10 M-1, and the binding energy increases by about 2.1 kcal mol-1 compared to p-OH Hoechst. The value of Ka determined at equilibrium equaled that determined from the ratio of association and dissociation rate constants from stopped-flow studies. The increase in affinity of the monohydroxy Hoechst analogue (m-OH) may originate from water-mediated hydrogen bonding with the minor groove. The further increase in affinity ... [truncated at 150 words]
Breusegem SY, Sadat-Ebrahimi SE, Douglas KT, Clegg RM, Loontiens FG.
Increased stability and lifetime of the complex formed between DNA and meta-phenyl-substituted Hoechst dyes as studied by fluorescence titrations and stopped-flow kinetics.
J Mol Biol. 2001; 308(4): 649-63.The large increase in fluorescence upon binding of five para- and meta-phenyl substituted hydroxy and methoxy derivatives of the Hoechst dye with poly[d(A-T)], d(CGCGAATTCGCG)2, and its corresponding T4-looped 28-mer hairpin was used to monitor the binding by equilibrium titrations and by stopped-flow kinetics. The affinity increases in the same order for the three DNAs: p-OH<m-OCH3, p-OH<m-OH<m-OH, p-OCH3<bis-m-OH. The association constants K(a) are three to 11 times larger for the AATT site than for poly[d(A-T)]. The AATT site binds m-OH Hoechst with K(a)=3.8 x 10(9 )M(-1) and bis-m-OH Hoechst with K(a)=1.9 x 10(10 )M(-1), which are seven and 37 times higher than p-OH Hoechst (Hoechst 33258), respectively. The high K(a )values determined at equilibrium agree with the kinetically defined association constants K(kin)=k(on)/k(off). The association-rate parameters k(on) were obtained by stopped-flow kinetics and the dissociation-rate parameters k(off) by dissociation kinetics using poly[d(A-5BrU)]. For binding to the AATT site, k(on) values are similar ... [truncated at 150 words]
Breusegem SY, Barry NP, Mackinnon AC, Williams BD, Clegg RM.
Integrin interactions in C. Elegans muscle studied in vivo by fluorescence resonance energy transfer (FRET).
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 162a, 656.2-Pos.Fluorescence microscopy was used to study integrin interactions in vivo in the nematode C. elegans. Integrins are important transmembrane receptor proteins involved in cell signaling and adhesion. In C. elegans muscle, dense bodies and M-lines anchor the myofilament lattice to the cell membrane and the extracellular matrix. These highly organized structures contain integrin heterodimers as well as cytoskeletal adapter proteins and signaling molecules. They are analogous to the focal adhesion complexes found in human cells and as such they are good systems to investigate integrin’s adhesive and cell signaling functions. Fluorescence resonance energy transfer (FRET) between fusion proteins with GFP mutants of different color allows the detection of direct intermolecular interactions in vivo. Transgenic animals were created in which protein pairs in the dense bodies or M-lines are labeled with cyan fluorescent protein (CFP) as a donor and yellow fluorescent protein (YFP) as an acceptor. Several methods were then used ... [truncated at 150 words]
Gohlke C, Holub O, Clegg RM.
FliFast: Software for fast fluorescence lifetime-resolved image acquisition with concurrent analysis and visual feedback.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 169a, 656.54-Pos.Fluorescence lifetime-resolved imaging FLI provides indispensable, valuable information than is not available from steady-state fluorescence image measurements. In general, because of the complexity of the analysis compared to the display of simple intensity images, FLI measurements have required much longer times to process and display. However, many biological and medical applications using fluorescence imaging require real time acquisition, processing and display. Real-time operation is necessary for following dynamic biological events, or carrying out medical diagnostics. We have developed a real-time FLI system for a variety of imaging applications. The instrument uses rapid frequency-domain data acquisition hardware; however, here we demonstrate software that specifically enables the real-time processing and highly informative, convenient and easy-to-understand display of the lifetime-resolved fluorescence information. A menu of possibilities is provided to the operator to assist in the on-line interpretation and control of real-time experiments and rapid events. The programs have been streamlined in order to ... [truncated at 150 words]
Holub O, Seufferheld MJ, Gohlke C, Govindjee, Clegg RM.
Application of real-time fluorescence lifetime-resolved imaging in photosynthesis: studies of maize leaves (Zea Mays), small mustard leaves (Arabidopsis Thaliana), and of individual wild-type and mutant cells of the green alga Chlamydomonas Reinhardtii.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 428a, 1819-Pos.Chlorophyll (Chl) a fluorescence from leaves of Zea Mays, Arabidopsis Thaliana and from single cells of wild type (WT) and non-photochemical quenching mutants (NPQ1 and NPQ2) of C. Reinhardtii was observed with a new high-speed instrument for measuring fluorescence lifetime-resolved microscope images. Objects are imaged using the frequency domain phase and modulation technique in homodyne mode. The laser light is modulated at a high frequency. The fluorescence image is focused onto a modulated image intensifier and the phase-resolved images are captured on a fast CCD camera, sequentially transferred to a PC computer and displayed in real-time. Rapid measurements are necessary during the fluorescence transient maximum (the P-level) following initial illumination. The observed lifetime heterogeneity is in accordance with photosynthetic functionality. The NPQ1 and NPQ2 mutants (Niyogi et al., 1997) used in this work accumulate violaxanthin and zeaxanthin, respectively. A decrease in the lifetime of Chl a in NPQ2 was observed ... [truncated at 150 words]
Majumdar ZK, D’Amico ML, Clegg RM.
Fluorescent Properties of Cy5 (5-N-N’-diethyltetramethylindodicarbocyanine) covalently linked to DNA.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 483a, 2066-Pos.Fluorescent dyes covalently linked at specific sites on nucleic acids are used for determining various properties of the nucleic acids, such as their structure and stability by the use of FRET. Photophysical studies lead to a better understanding of the interactions of molecules with the chromophore. The Cy3-Cy5 pair is an optimal donor-acceptor pair for FRET. Both dyes have high extinction coefficients (150,000 and 250,000 cm^-1M^-1, respectively) and their emission spectra are well separated from most sample autofluorescence. We present an analysis of the fluorescence properties of Cy5 attached to the 5’ end of double-stranded (ds) DNA, with a C3-linker to the phosphodiester. Measurements of the fluorescence lifetime, time-resolved anisotropy and steady state measurements are used to understand the structure of the Cy5-DNA complex. Temperature and solvent effects on the fluorescence properties are studied as well. In addition to providing a better understanding of the structure of the Cy5-DNA complex, ... [truncated at 150 words]
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Conformational behavior of DNA-Cy3 complex.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 483a, 2065-Pos.The photophysical properties of 5-N,N’-diethyl-tetramethylindocarbocyanine (Cy3) attached to the blunt end of an 18bp deoxyoligonucleotides have previously been studied. We now extend the investigation by incorporating an extra base which intervenes the dye molecule and blunt end of the 18bp deoxyoligonucleotides. The dye molecule is attached via a C3 linker to the photodiester on the strand with extra nucleotide. Steady-state flourescence, life time and anisotropy decay are measured under various temperature and solvent conditions. The results are compared with earlier work to provide better understand of the conformational behavior of DNA-Cy3 complex.
Breusegem SY, Loontiens FG, Regenfuss P, Clegg RM.
Kinetics of binding of Hoechst dyes to DNA studied by stopped-flow fluorescence techniques.
Drug-Nucleic Acid Interactions (Methods in Enzymology, Vol. 340). Academic Press, pp. 212-33, 2001. ISBN: 9780121822415
Holub O, Seufferheld MJ, Gohlke C, Govindjee, Clegg RM.
Fluorescence lifetime imaging (FLI) in real-time - a new technique in photosynthesis research.
Photosynthetica. 2000; 8(4): 581-599.We describe an instrument that allows the rapid measurement of fluorescence lifetime-resolved images of leaves as well as sub-cellular structures of intact plants or single cells of algae. Lifetime and intensity fluorescence images can be acquired and displayed in real time (up to 55 lifetime-resolved images per s). Our imaging technique therefore allows rapid measurements that are necessary to determine the fluorescence lifetimes at the maximum (P level) fluorescence following initial illumination during the chlorophyll (Chl) a fluorescence transient (induction) in photosynthetic organisms. We demonstrate the application of this new instrument and methodology to measurements of: (1) Arabidopsis thaliana leaves showing the effect of dehydration on the fluorescence lifetime images; (2) Zea mays leaves showing differences in the fluorescence lifetimes due to differences in the bundle sheath cells (having a higher amount of low yield photosystem 1) and the mesophyll cells (having a higher amount of high yield photosystem 2); ... [truncated at 150 words]
Stühmeier F, Hillisch A, Clegg RM, Diekmann S.
Fluorescence energy transfer analysis of DNA structures containing several bulges and their interaction with CAP.
J Mol Biol. 2000; 302(5): 1081-100.DNA molecules with three bulges separated by double-stranded helical sections of B-DNA were constructed to be used as substrates for DNA-protein binding assays. Fluorescence resonance energy transfer (FRET) between dye molecules attached to the 5'-ends of the DNA molecules is used to monitor the protein binding. The A5 bulge, which consists of five unpaired adenine nucleotides, alters the direction of the helical axis by approximately 80 to 90 at every bulge site. Computer molecular modeling facilitated a pre-selection of suitable helix lengths that bring the labeled ends of the three-bulge DNA molecules (60 to 70 base-pairs long) into close proximity. The FRET experiments verified that the labeled ends of the helices of these long molecules were indeed close. A series of FRET experiments was carried out with two A5 and two A7 bulge molecules. The relative positions of the bulges were varied along the central helical DNA sequence (between ... [truncated at 150 words]
Stühmeier F, Clegg RM, Hillisch A, Diekmann S.
Practical aspects of fluorescence resonance energy transfer (FRET) and its applications in nucleic acid biochemistry.
DNA-Protein Interactions: A Practical Approach. By A Travers and M Buckle (Editors). Oxford University Press, pp. 77-94, 2000. ISBN: 9780199636914Clegg RM, Stühmeier F, Gohlke C, Vámosi G.
Conformations and stabilities of nucleic acids using FRET.
Abstr Paper Am Chem Soc Natl Meet. 2000; 220: U239-U239 568-PHYS Part 2.Holub O, Seufferheld MJ, Gohlke C, Clegg RM, Govindjee.
Real-time fluorescence lifetime-resolved images of individual cells of wild type and NPQ mutants of Chlamydomonas reinhardtii.
9th International Conference on the Cell and Molecular Biology of Chlamydamonas, Amsterdam, The Netherlands, May 21-26, 2000.
The chlorophyll a (Chl a) fluorescence from single cells of wild type (WT) and non-photochemical quenching (NPQ) mutants (NPQ1 and NPQ2) of Chlamydomonas reinhardtii was observed with a new high-speed instrument for measuring fluorescence lifetime-resolved images (see Instrumentation). The mutants NPQ1 and NPQ2 used in this work (Niyogi et al., 1997) , accumulate violaxanthin and zeaxanthin, respectively. Fluorescence transient studies (see poster by Seufferheld et al., 2000) show strong quenching of Chl a fluorescence in the NPQ2 mutant in comparison to the WT and the NPQ1 mutant. If the quenching is due to Förster transfer from Chl a to zeaxanthin, then the lifetime of Chl a in the NPQ2 mutant will decrease. The time-resolved fluorescence microscopy of the NPQ mutants can therefore be used to study the role of the Xanthophyll cycle in the non-photochemical quenching process in single cells. This work is supported by NIH PHS 5 P41-RRO3155 and ... [truncated at 150 words]
Holub O, Gohlke C, Clegg RM.
Want to know something about your fluorescent samples no lenses can resolve? Big scale scanning setup for real-time fluorescence lifetime-resolved imaging.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 1464-Pos.We present a new high-speed setup for measuring fluorescence lifetime-resolved images of larger objects. This instrument allows scanning of extended objects using the phase and modulation technique in homodyne mode. A low-resolution objective allows the laser illumination of the sample placed on a xy-scanning stage and also captures the whole fluorescent image and projects it on a modulated image intensifier. A fast CCD camera captures the incrementally phase-delayed modulated inages, which are then processed and displayed in real-time by programs on a PC computer. Light modulation (CW laser) is achieved by the use of a standing wave acousto-optical modulator. We demonstrate the functionality and use of the instrument with different examples. This work is supported by NIH PHS 5 P41-RRO3155 and NSF DBI96-0240.
Hanson KM, Barry NP, Gratton E, Clegg RM.
Fluorescence lifetime imaging of pH in the stratum corneum.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 1478-Pos.Fluorescence lifetime imaging microscopy (FLIM) is being developed to explore the pH gradient in the uppermost layer of the skin's epidermis, the stratum corneum (SC). The SC provides the primary barrier between the environment and the body's interior. It is also considered to play an essential role in normal skin renewal each month (cornification). Over the extremely thin stratum corneum (-20 pm) the pH has been reported to gradually change from 4-5.6 at the skin's surface to pH 7.2 at the stratum corneum-granulosumjunction. This corresponds to an increase of up to 1000-fold in the proton concentration over the 20-40 SC cellular layers. Tape-stripping measurements have been employed to study pH changes in the SC; however, such measurements are invasive and provide little information on the location and shape of the proposed SC pH gradient. Consequently, we are developing FLIM experiments to explore non-invasively pH changes within the heterogeneous tissue. For ... [truncated at 150 words]
Breusegem SY, Loontiens FG, Clegg RM.
Binding of Hoechst 33258 and DAPI to (A/T)4 DNA sites: base-sequence specificity determined by fluorescence titrations and stopped flow kinetics.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 1804-Pos.Hoechst 33258 (a bis-benzimidazole compound) and DAPI (4',6'-diamidino-2-phenylindole) become brightly fluorescent when binding in the minor groove of DNA, preferentially at respectively 5 or 3 contiguous AT-base pairs. We determined the binding affinity at 20"C of both dyes for isolated (A/T)4-sites incorporated in a 28-nucleotide-hairpin by fluorescence titrations. Because of the high affinity the titrations had to be carried out at very low dye concentrations of -2 nM. The affinities of DAPI and Hoechst 33258 for the AATT site are similar (e.g. Hoechst 33258 association constant = 5.2*108 M') and the affinity decrease in the sequence series runs parallel for both dyes: AATT>TAAT>ATAT>TATAzTTAA for Hoechst 33258 and AATT>TAATnATAT>TATAzsTTAA for DAPI. For Hoechst 33258 the association constants for the most and the least affine site in the series differ by a factor of 190, for DAPI this difference is only a factor of 30. The binding kinetics of Hoechst 33258 were ... [truncated at 150 words]
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A glimpse of nucleic acid conformations through the eyes of fluorescence.
4th International Weber Symposium. Maui, Hawaii. June 23-27, 1999.
Fluorescence experiments have been applied more frequently in the last years for investigating many different facets of nucleic acid structures. In addition to straightforward surveillance applications (e.g. binding assays and identification of the presence of particular components), and quantitative determinations of relative amounts of nucleic acids, fluorescence is invaluable as a probe of DNA/RNA structure and dynamics. It is uniquely applicable to many different experimental situations, from low solution concentrations and single molecule experiments, to very complex structures such as seen in chromosomes and ribozymes, in solution experiments as well as in the fluorescence microscope. The exceptionally broad applicability of fluorescence techniques, coupled with the versatile opportunities offered by fluorescence for determining detailed physical information related to the molecular state of the nucleic acids, as well as providing very sensitive detection opportunities for molecular assays, makes fluorescence a uniquely invaluable tool with remarkable information content. In this seminar several general ... [truncated at 150 words]
Breusegem SY, Loontiens FG, Clegg RM.
Binding kinetics of Hoechst 33258 and some of its derivatives to DNA.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).The dye Hoechst 33258 and some of its meta-phenyl-substituted derivatives bind in the minor groove of B-DNA to sequences consisting of 4 to 5 juxtaposed A or T bases. The intense increase in dye fluorescence upon binding was used to monitor the binding equilibrium and the stopped-flow kinetics. The binding reaction was measured with polymeric DNA's (CT-DNA, poly[d(A-T)] and poly[d(A-5BrU)]), the duplex d(CGCGAATTCGCG)2, and with a dilution-resistant 28-nucleotide-hairpin in which the G-3' and C-5' ends of the duplex are connected by a tetra-T sequence. Low concentrations of the dye (1 to 5 nM) were used to prevent its stoichiometric associations on polymeric DNA. DNA concentrations were expressed as sites for the dye. The kinetics for binding of Hoechst 33258 were consistent with a single-step bimolecular association that is practically controlled by diffusion: for calf-thymus DNA, k+=1.5x109 M-1 sec-1; with the other DNA's, somewhat smaller k1 values were obtained. Kinetically defined ... [truncated at 150 words]
Stühmeier F, Hillisch A, Diekmann S, Clegg RM.
Fluorescence resonance energy transfer analysis of DNS-structures containing several A5 bulges and their interaction with proteins.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).Fluorescence Resonance Energy Transfer (FRET) was utilized to probe the geometry of DNA structures containing multiple A5 bulges. The bulges (unpaired nucleotides in one strand) fold back the molecule and decrease considerably the distance between the ends of the DNA; therefore, energy transfer could be detected between two dyes covalently attached to the 5'-ends of the DNA, although the dyes were separated by up to 60 to 70 consecutive bp. The molecules contain 24 bp long sequence with the binding site for the DNA binding protein CAP, which bends the helix of the DNA. The structural distortion of the DNA under the influence of CAP was monitored by measuring the change of the FRET efficiency between the dyes attached to the DNA. No direct interaction between CAP and the dyes could be detected. A quantitative comparison between the FRET data and molecular models based on the NMR structure of the ... [truncated at 150 words]
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"Second order" sequence specificity of DAPI-binding to AT-regions in DNA.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).DAPI binds strongly (with pronounced sequence specific affinities in the nanomolar range [Loontiens et al., 1991]) in the minor groove to AT-rich regions of DNA. We measured the binding constants to "AATT" and "TTAA"-containing oligonucleotides of different lengths in a study involving fluorescence energy transfer between DAPI molecules bound to oligonucleotides with these two sequences separated by different lengths of helix, and found a significant sequence dependence of the binding affinities. Time-resolved fluorescence measurements of DAPI bound to oligonucleotides containing only one of these binding sites showed only lifetimes between 0.2-4 ns at all dye/DNA ratios for 5'CGCGAATTCGCG3', but an additional lifetime about 10 ns appeared at larger dye/DNA ratio (where the affine sites were already occupied) when DAPI interacts with 5'CGCGTTAACGCG3' and 5'CGAATTGGCACAGC3'. This longer lifetime has also been observed with other repetitive sequences of DNA (poly d[AT] and d[GC]) and CT-DNA [Cavatorta et al., Biophys. Chem. 22 (1985)]. ... [truncated at 150 words]
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The helix-coil transition of DNA duplexes and hairpins observed by multiple fluorescence parameters.
Biochemistry. 1998; 37(40): 14300-16.The thermal denaturation of 8-20-bp DNA duplexes labeled with fluorescein and tetramethylrhodamine at opposing 5'-ends was investigated by monitoring the fluorescence intensity of the dyes, the fluorescence anisotropy of tetramethylrhodamine, the fluorescence resonance energy transfer between fluorescein and rhodamine, and, for the 20-bp duplex, the UV absorption. Melting experiments with the single strands of the duplexes revealed that the single strands can form hairpins stabilized by only a few base pairs. The thermal denaturation curves of the duplexes were fitted well to an extended all-or-none model assuming that only the fully base-paired duplex, the maximally base-paired hairpin, and the random coil conformations are present simultaneously. The extent-of-melting versus temperature curves derived from the different spectroscopic parameters are nearly identical, provided that the analysis of the baselines is carried out correctly; the DeltaH and DeltaS of the dissociation compare well with predictions based on nearest neighbor interaction values available in the ... [truncated at 150 words]
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Reversible stalling of transcription elongation complexes by high pressure.
Biophys J. 1998; 75(1): 453-62. PMCID: PMC1299718We have investigated the effect of high hydrostatic pressure on the stability of RNA polymerase molecules during transcription. RNA polymerase molecules participating in stalled or active ternary transcribing complexes do not dissociate from the template DNA and nascent RNA at pressures up to 180 MPa. A lower limit for the free energy of stabilization of an elongating ternary complex relative to the quaternary structure of the free RNAP molecules is estimated to be 20 kcal/mol. The rate of elongation decreases at high pressure; transcription completely halts at sufficiently high pressure. The overall rate of elongation has an apparent activation volume (DeltaVdouble dagger) of 55-65 ml . mol-1 (at 35 degrees C). The pressure-stalled transcripts are stable and resume elongation at the prepressure rate upon decompression. The efficiency of termination decreases at the rho-independent terminator tR2 after the transcription reaction has been exposed to high pressure. This suggests that high pressure ... [truncated at 150 words]
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Stereochemistry of DNA molecules by homo-energy transfer.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2): A288, W-Pos192.Förster Fluorescence Resonance Energy Transfer (FRET) between two identical dyes has been used for studying the stereochemistry of complex DNA molecules. The dye DAPI binds to DNA with a tight site-selective affinity, the crystal structure of dve-DNA complexes is known. Oligonucleotides were made with two binding sites, 5' AATT 3' and a 5' TTAA 3' separated by varying lengths of duplex DNA. The efficiency of energy transfer was determined from fluorescence anisotropy, which decreases as the energy is transferred between the two identical dye molecules (the intensity of fluorescence is not affected by this homo-transfer process). The two binding sites have different binding affinities: the binding affinities were determined simultaneously with the efficiency of energy transfer. The fluorescence anisotropies were determined during a continuous automatic titration of the dye to the DNA. The binding of DAPI to the double-stranded oligonucleotides was analyzed with a model consisting of three binding processes: ... [truncated at 150 words]
Schneider PC, Holub O, Clegg RM.
Real-time fluorescence lifetime-resolved images of 5-aminolevulinic acid (ALA)-induced Protoporphyrin IX (PPIX) in cells.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2), Tu-Pos250.An instrument using the phase and modulation technique in homodyne mode was developed that is capable of acquiring, processing and displaying fluorescence lifetime-resolved images in real-time (frame refresh rates up to 13 Hz). Using fluorescence lifetime-resolved imaging microscopy (FLIM) luminescence emission can be lifetime-resolved directly) at each location of a microscope image. With this method the mean lifetime is determined simultaneously at every pixel of a digital image. A SUN UltraSparc Computer with Creator 3D graphics processor is used. Programs for data acquisition. image analysis and 3D display modes have been written using AVS/express. We observed PPIX accumulation in cells - fast data acquisition is important due to the fast bleaching rate. In the biosynthetic pathway of heme. ALA is converted to PPIX. an endogenous fluorescent photosensitizer. A surplus of ALA leads to an accumulation of PPIX because of a strong heme feedback mechanism. This was studied in several cell ... [truncated at 150 words]
Bassi GS, Murchie AIH, Walter F, Clegg RM, Lilley DMJ.
Ion-induced folding of the hammerhead ribozyme: a fluorescence resonance energy transfer study.
EMBO J. 1997; 16(24): 7481-9.The ion-induced folding transitions of the hammerhead ribozyme have been analysed by fluorescence resonance energy transfer. The hammerhead ribozyme may be regarded as a special example of a three-way RNA junction, the global structure of which has been studied by comparing the distances (as energy transfer efficiencies) between the ends of pairs of labelled arms for the three possible end-to-end vectors as a function of magnesium ion concentration. The data support two sequential ion-dependent transitions, which can be interpreted in the light of the crystal structures of the hammerhead ribozyme. The first transition corresponds to the formation of a coaxial stacking between helices II and III; the data can be fully explained by a model in which the transition is induced by a single magnesium ion which binds with an apparent association constant of 8000-10000 M-1. The second structural transition corresponds to the formation of the catalytic domain of the ... [truncated at 150 words]
Stühmeier F, Lilley DMJ, Clegg RM.
Effect of additional unpaired bases on the stability of three-way DNA junctions studied by fluorescence techniques.
Biochemistry. 1997; 36(44): 13539-51.Fluorescence melting experiments were carried out to determine the relative stability of three-way DNA junctions with and without extrahelical adenine nucleotides in one strand at the branch point of the junction (i.e., An bulges where n = 0, 1, 2, and 3). The oligonucleotides were labeled with chromophores at the 5' ends of the strands. The progress of the thermal denaturation was followed by monitoring the fluorescence intensities and anisotropies of the dyes and the fluorescence resonance energy transfer between the two dyes. The results of the thermal denaturation experiments are interpreted and discussed in terms of either two-state thermodynamic models or statistical models for the thermal denaturation. The junctions all melt at the same temperature (at equal concentrations) within the error of the Tm determination, regardless of the presence, or absence, of the bulge. It is suggested that the denaturation of the helical arms begins primarily at the free ... [truncated at 150 words]
Stühmeier F, Welch JB, Murchie AIH, Lilley DMJ, Clegg RM.
Global structure of three-way DNA junctions with and without additional unpaired bases: a fluorescence resonance energy transfer analysis.
Biochemistry. 1997; 36(44): 13530-8.The structure of three-way DNA junctions with and without extrahelical adenine nucleotides in one strand at the branch point of the junction (i.e., An bulges with n = 0, 1, 2, and 3) has been investigated by fluorescence resonance energy transfer. The structure of the junction without bulged nucleotides was found to have a symmetric trigonal geometry. With bulges, the arrangement of the arms becomes asymmetrical. The energy transfer results suggest a model of bulged junctions where the angle between two of the arms is significantly smaller than between the other two pairs of arms. The acute angle becomes smaller as the number of nucleotides in the bulge increases. The FRET efficiencies of the junctions are the same in the presence of Mg++ and Na+ ions.
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Rapid acquisition, analysis, and display of fluorescence lifetime-resolved images for real-time applications.
Rev Sci Instrum. 1997; 68(11): 4107-4119.Fluorescence lifetime-resolved imaging (FLI) is a relatively new technique of fluorescence imaging whereby the spatial distribution of fluorescence decay times can be determined directly at every pixel of an image simultaneously. The fluorescence decay times of many chromophores can act as sensitive gauges of their molecular environments. By employing measurement techniques that are quantitatively related to the radiative dynamics of the dye molecules (in the nanosecond time range), additional physical parameters are available for discerning different fluorophores with disparate lifetimes, or for characterizing a single fluorophore in different surroundings. Many physical processes such as molecular aggregation, binding of dyes to macromolecular species, inclusion of chromophores in specific cellular organwelles, fluorescence resonance energy transfer, and dynamic quenching determine the excited-state lifetime of a fluorophore. The FLI technique provides a way to measure these processes directly at 103–106 pixels in an image. In addition, if image domains differ with respect to the ... [truncated at 150 words]
Duckett DR, Murchie AIH, Clegg RM, Bassi GS, Giraud-Panis MJE, Lilley DMJ.
Nucleic acid structure and recognition.
Biophys Chem. 1997; 68(1-3): 53-62.We review the global structures adopted by branched nucleic acids, including three- and four-way helical junctions in DNA and RNA. We find that some general folding principles emerge. First, all the structures exhibit a tendency to undergo pairwise coaxial helical stacking when permitted by the local stereochemistry of strand exchange. Second, metal ions generally play an important role in facilitating folding of branched nucleic acids. These principles can be applied to functionally important branched nucleic acids, such as the Holliday DNA junction of genetic recombination, and the hammerhead ribozyme in RNA.
Schneider PC, Holub O, Clegg RM.
Real-time fluorescence lifetime-resolved imaging.
Second Conference on Fluorescence Microscopy & Fluorescent Probes. April 9-12, 1997. Prague, Czechia.
We present a new instrument based on a SUN UltraSparc Computer with Creator 3D graphics processor that is capable of acquiring, processing and displaying fluorescence lifetime-resolved images at a very high speed. The frame refresh rate is dependent upon the available amount of light, the data acquisition mode, the algorithms used for processing data and the sophistication of the display. Refresh rates of up to 15 Hz have been achieved, although refresh rates of 1-7 Hz are typical. The system uses the phase and modulation technique in homodyne mode. According to the operator's requirements several methods of data processing can be applied to yield different information. Using an additional photomultiplier non-imaging lifetime measurements in a microscope are also possible. The programs, including hardware control, have been written using AVS/express with newly developed C modules. We show examples from three major fields of applications: real-time FLIM, rapid fluorescence lifetime micro assays ... [truncated at 150 words]
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Stereochemistry of DNA molecules by homo-energy transfer.
Second Conference on Fluorescence Microscopy & Fluorescent Probes. April 9-12, 1997. Prague, Czechia.
We used the orientational dependence of Förster Fluorescence Resonance Energy Transfer (FRET) between two identical fluorescent dyes to develop a sensitive fluorescence assay for studying stereochemical geometries of complex DNA molecules. The dye DAPI was chosen for this, because it binds with a high affinity in a site selective manner to DNA and the crystal structure of dye-DNA complexes have been reported. To calibrate the method, and show the applicability, a series of oligonucleotides was constructed with two binding sites, 5' AATT 3' and a 5' TTAA 3', separated by varying lengths of duplex DNA. The efficiency of energy transfer was determined by measuring the fluorescence anisotropy, which decreases as the energy is transferred between the two identical dye molecules (the intensity of fluorescence is not affected by this homo-transfer process). The two binding sites were found to have different binding affinities, and this made it necessary to determine the ... [truncated at 150 words]
Erijman L, Villas-Boas M, Clegg RM.
The effect of high pressure on protein-DNA interactions: nucleosomes and RNA polymerase enzymatic activity.
22nd Annual Lorne Conference On Protein Structure And Function. February 9-13, 1997. Lorne, Australia.
High pressure studies of two multisubunit protein-DNA systems will be presented: nucleosomes and E. coli RNA polymerase. The application of a moderate level of high pressure (<3000 atmospheres) is unique in its propensity to affect primarily the state of aggregation of multisubunit proteins, and hydrostatic pressure is a convenient and opportune method for modifying the conformations and subunit-subunit interactions of proteins. Multisubunit protein complexes can often be dissociated by high pressure into their constituent subunits; complexes between regulatory proteins and their specific ligands and cognate DNA sites are affected as well.
High pressure studies have resulted in several important conclusions concerning the structures and functions of nucleosomes: 1) the free energy difference ∆G between two major populations of nucleosome structures present at physiological conditions is only ~6-8 kcal/mole nucleosome; 2) the apparent volume change ∆V between these two populations of conformations is ~55 ml/mole nucleosome; 3) the number of ions associated ... [truncated at 150 words]
Clegg RM, Schneider PC, Jovin TM.
Fluorescence lifetime-resolved imaging microscopy.
Biomedical Optical Instrumentation and Laser-assisted Biotechnology. Erice, Italy. Nov 10-22, 1995.
Biomedical Optical Instrumentation and Laser-assisted Biotechnology (NATO Science Series E, Vol. 325). By AMV Scheggi, S Martellucci, AN Chester, and R Pratesi (Editors). Kluwer Academic Press, pp. 143-156, 1996. ISBN: 0792341724FLIM - "Fluorescence Lifetime-resolved Imaging Microscopy" - is a relatively new fluorescence imaging technique by which the temporal attributes of luminescence emission can be measured directly at each location of a microscope image. In the frequency-domain FLIM technique described here nanosecond fluorescence decay characteristics can be quantified simultaneously at every picture element (pixel) of a digital electronic image. The mean lifetime of the emission can be determined with a high spatial and temporal resolution and different fluorescence components with differing decay times can be enhanced or suppressed throughout a fluorescence image. For references see [1] and see in the instrumentation section below.
The most common application of fluorescence microscopy is to display the spatial localization of a fluorescence probe, or of fluorescently labeled macromolecules, in a microscopic specimen. More recently there has been increased interest to derive more detailed information concerning the physical and chemical environment of the fluorescent probes, as ... [truncated at 150 words]
Vámosi G, Gohlke C, Clegg RM.
Photophysics of 5-carboxytetramethylrhodamine linked to the 5'-end of ss & ds DNA molecules.
XIIth International Biophysics Congress, Amsterdam, The Netherlands, August 11-16, 1996.
Prog Biophys Mol Biol. 1996; 65(Suppl 1): 76, P-B1-31.Purpose: To determine the photophysical properties of tetramethylrhodamine (TMRh) conjugated to single-stranded (ss) and doublestranded (ds) DNA oligomers.
Methods: TMRh was coupled (via a C6 linker) to the 5’ end of oligonucleotides. Fluorescence measurements on ds and ss TMRh-labeled DNA species were made over a large range of NaCl concentrations and temperatures, and in the presence of added ethanol. Static and lifetime-resolved fluorescence results are presented.
Results: Three well-defined states of TMRh-DNA exist at lower (cl00 mM) and at higher ionic strengths. Two states fluoresce with lifetimes of 0.5 - 1.5 ns and 2.5 - 4.5 ns; a third state does not fluoresce (dark species). All three states of TMRh are present on ds and ss DNA. It is possible to extract thermodynamic parameters related to the interaction of the TMRb with the nucleic acid molecule, and of ions with the TMRh-DNA complex.
Conclusions: Three states of TMRh conjugated to DNA are present. ... [truncated at 150 words]
Stühmeier F, Welch JB, Lilley DMJ, Clegg RM.
Fluorescence measurements of DNA three-way junctions.
XIIth International Biophysics Congress, Amsterdam, The Netherlands, August 11-16, 1996.
Prog Biophys Mol Biol. 1996; 65(Suppl 1): 131, P-B1-29.Purpose: The influence of 1, 2 and 3 unpaired adenine-nucleotides (bulges) at the branch point
of DNA three-way junctions on the relative orientation of the arms of the molecule has been investigated.
Methods: A series of fluorescence energy transfer efficiencies were determined between dyes attached (5’-end labeling) to the three different pairs of arms of DNA three-way junctions. The thermal denaturation was also investigated using fluorescence spectroscopy.
Results: As the number of unpaired nucleotides in the bulge increases, the FRET efficiencies between two pairs of labeled arms decrease only slightly; the FRET-efficiency for one labeled pair of arms strongly increases. Rbodamine shows an apparent sequence-specific quenching of the fluorescence in the single-stranded region.
Conclusions: The molecules without a bulge adopt a structure with approximately equal distances between the 5’ ends of the molecule. The stereochemical arrangement of the arms relative to a 1, 2 or 3 bulge is different; this non-symmetrical structure will also ... [truncated at 150 words]
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Transcriptional inhibition by high pressure in vitro.
XIIth International Biophysics Congress, Amsterdam, The Netherlands, August 11-16, 1996.
Prog Biophys Mol Biol. 1996; 65(Suppl 1): 82, P-B2-10.Purpose: E. coli RNA Polymerase undergoes cycles of sequence-dependent conformational changes in transcriptional complexes during RNA synthesis. Many mechanistic features remain unclear. We have used high pressure methods to investigate the stability and flexibility of elongating ternary complexes.
Methods: Radioactivity and fluorescence assays were employed to examine the pressure stability and activity of RNAP participating in the elongation phase of transcription.
Results: The rate of elongation by E. coli RNAP on DNA templates can be halted reversibly at high pressures. Analysis of solvent and temperature dependence of the pressure-induced inhibition shows evidence for major conformational changes in the core polymerase enzyme during elongation.
Conclusions: Pressure perturbs nucleic acid-protein and protein-protein interactions, providing insight into the mechanism of transcription. We discuss our results in the framework of both the nucleic acid destabilization model and the “inchworm” elongation model.
Vámosi G, Gohlke C, Clegg RM.
Fluorescence characteristics of 5-carboxytetramethylrhodamine linked covalently to the 5' end of oligonucleotides: multiple conformers of single-stranded and double-stranded dye-DNA complexes.
Biophys J. 1996; 71(2): 972-94. PMCID: PMC1233554Fluorescence steady-state and lifetime experiments have been carried out on duplex and single-stranded DNA molecules labeled at the 5' ends with 5-carboxytetramethylrhodamine (TMRh). The temperature and ionic strength of the solutions were varied over large ranges. The results reveal at least three well-defined states of the TMRh-DNA molecules for the single-stranded as well as for the double-stranded DNA molecules. Two states are fluorescent, with lifetimes in the range of 0.5-1 ns and 2.5-3 ns. A third state of TMRh-DNA does not fluoresce (a dark species of TMRh-DNA). The distribution of the TMRh-DNA molecules among these three states is strongly temperature and ionic strength dependent. Estimates are made of some reaction parameters of the multistate model. The results are discussed in terms of the photophysics of TMRh, and consequences of the multiple conformers of TMRh-DNA for studies involving fluorescence studies with TMRh-labeled DNA are considered.
Schneider PC, Gadella TWJ Jr, Jovin TM, Clegg RM.
Analytica. April 23-25 1996, Munich, Germany.
FLIM is a relatively new fluorescence imaging technique by which the temporal attributes of luminescence emission from a fluorescence microscope image can be measured directly at each location of the microscope image. By using the FLIM technique the nanosecond fluorescence decay characteristics can be quantified at every pixel of the digital electronic image simultaneously. It is possible to measure directly the mean lifetime of the emission from a spatially resolved fluorescence image and to discriminate among several fluorescence components in a fluorescence image based on their differing decay times.
An analysis of the decay profile of fluorescence emission provides valuable information concerning fundamental physical and chemical processes that occur while the molecules are in the excited state, e.g. quenching processes, energy transfer, the rate of intersystem crossing from the excited singlet to the triplet state, excited-state reactions, excited state complexes, diffusion processes, molecular rotation, etc.. Steady-state fluorescence arises is an ... [truncated at 150 words]
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Fluorescence resonance energy transfer.
Fluorescence Imaging Spectroscopy and Microscopy (Chemical Analysis: A Series of Monographs on Analytical Chemistry and Its Applications). By XF Wang and B Herman (Editors). John Wiley & Sons, pp. 179-252, 1996. ISBN: 9780471015277
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High pressure electrophoresis in narrow bore glass tubes: one- and two-dimensional separations of protein subunits.
Rev Sci Instrum. 1996; 67(3): 813-817.A mini-gel tube electrophoresis apparatus that is easily constructed and simple to operate has been developed. The system can be accommodated in standard commercially available high pressure tubing, and has been tested at up to 200 MPa. The narrow diameter of the glass tubes allows rapid and efficient dissipation of heat. Adequate buffer capacity is maintained in the low volume anode reservoir by increasing the concentration of the buffer. Analytical separations can be achieved in short times with high resolution. After the electrophoresis has been carried out at elevated pressure, the gel can easily be extruded from the tube and loaded onto a standard slab gel for a second-dimensional run at atmospheric pressure. We illustrate the application of this apparatus with the high pressure gel electrophoresis separation and subsequent identification of the constituent subunits of E. coli RNA polymerase.
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Fluorescence lifetime-resolved imaging microscopy: a general description of the lifetime-resolved imaging measurements.
Based on the proceedings of a conference held in Prague, Czech Republic, June 25-28, 1995.
Fluorescence Microscopy and Fluorescent Probes. By J Slavik (Editors). Plenum Press, New York, pp. 15-33, 1996. ISBN: 0306453924
Gadella TWJ Jr, van Hoek A, Visser AJWG, Schneider PC, Jovin TM, Clegg RM.
Frequency-domain fluorescence lifetime imaging microscopy: methodology and examples.
Scanning. 1996; 18(1): 59-60.Fluorescence lifetime imaging microscopy (FLIM) is a new technique for resolving nanosecond temporal characteristics of fluorophores in a spatially extended fluorescence image; the dynamic features are captured at every pixel of an image simultaneously. Various imaging applications, involving imaging biological specimens with multiple fluorophores, can benefit from this new technology. Our frequency domain instruments employ periodically modulated excitation light, synchronous modulation of the amplification stages of a microchannel plate (MCP) intensifier, and subsequent digital recording of the image with a slow-scan charge-coupled device (CCD) camera. The FLIM instruments can be operated such that the modulation frequency of the excitation light and the intensifier amplification are either slightly different (heterodyne mode) or the same (homodyne mode). The image data acquisition takes only a few seconds, depending on the intensity of the image and the number of images acquired. More detailed descriptions of our lifetime-resolved fluorescence imaging instruments are given elsewhere.1–3 Typical ... [truncated at 150 words]
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Heterogeneity of E. coli RNA polymerase revealed by high pressure.
J Mol Biol. 1995; 253(2): 259-65.The activity and subunit association of Escherichia coli RNA polymerase has been investigated by high pressure techniques (up to 2000 atm). The extent of subunit dissociation in the presence and absence of DNA was monitored by carrying out electrophoresis directly at elevated pressure. The degree of inactivation brought about by high pressure was determined by measuring the enzyme activity following decompression. The loss of activity if the enzyme molecules are not actively involved in transcription is correlated with the extent of association of the polymerase subunits. At any particular pressure only a fraction of polymerase molecules becomes inactivated; the remaining fraction retains its original activity characteristics. If the enzyme molecules are actively involved in transcription when the high pressure is applied, the RNA synthesis can be completely halted, but the elongation activity is fully recovered on decompression. The experimental results are consistent with the existence of a broad distribution of ... [truncated at 150 words]
Lilley DMJ, Clegg RM, Diekmann S, Seeman NC, von Kitzing E, Hagerman PJ.
A nomenclature of junctions and branchpoints in nucleic acids.
Nucleic Acids Res. 1995; 23(17): 3363-3364.Branchpoints are a common feature of folded nucleic acids. They may be created from double stranded DNA during processes generating sequence rearrangements such as homologous or site-specific recombination, or by the secondary and tertiary folding of single-stranded nucleic acids. The latter is especially important in RNA, and the branched structure of such molecules may be central to their function. The structures of branchpoints in DNA molecules are known to be recognised by a number of enzymes that may bind to and manipulate DNAjunctions. As the complexity of the junctions studied in laboratories has increased, a need has arisen to find a nomenclature that will allow the unambiguous description of any given branchpoint...
Arndt-Jovin DJ, Clegg RM, Jovin TM.
Intracellular pH and ion distributions in living cells determined by phase-detected fluorescence lifetime imaging, FLIM.
3rd International Weber Symposium. Maui, Hawaii. July 30-August 2, 1995.
Although it has been possible for some years to image intracellular pH and ion concentration distributions in living cells, these measurements usually require dual wavelength excitation and/or emission imaging and are complicated by variations in dye concentration in intracellular compartments. We have constructed a fluorescence lifetime imaging microscope (FLIM) with both high spatial and temporal resolution utilizing a frequency domain technique and homodyne phase detection. The light source for this microscope is a CW ion laser whose intensity is sinusoidally modulated at ~40 MHz by an AOM. The relative phase and modulation depth of the fluorescence emission is detected most efficiently by the homodyne technique. In our implementation, the optical gain of an image intensifier coupled to a slow-scan CCD camera is modulated by driving the high-voltage microchannel plate at the same frequency as the excitation but with systematic, incremental high frequency phase shifts for each image collected on the ... [truncated at 150 words]
Jurkiewicz E, Villas-Boas M, Silva JL, Weber G, Hunsmann G, Clegg RM.
Inactivation of simian immunodeficiency virus by hydrostatic pressure.
Proc Natl Acad Sci USA. 1995; 92(15): 6935-7. PMCID: PMC41445The inactivation of the simian immunodeficiency viruses SIVmac251 and SIVagm by pressures of 150 and 250 MPa was determined. The extent of inactivation depended on the time that the virus was subjected to compression as well as the level of the pressure and at 150 Mpa reached 5 log10 dilution units after approximately 10 hr. The inactivations, which were uniformly carried out at room temperature, were independent of the concentration of the virus. Possible applications of pressure inactivation for molecular biological and clinical use are discussed.
Lilley DMJ, Clegg RM, Diekmann S, Seeman NC, von Kitzing E, Hagerman PJ.
Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). A nomenclature of junctions and branchpoints in nucleic acids. Recommendations 1994.
Eur J Biochem. 1995; 230(1): 1-2.-
Fluorescence resonance energy transfer.
Curr Opin Biotechnol. 1995; 6(1): 103-110.In the past year, a number of studies have demonstrated the utility of fluorescence resonance energy transfer as a technique for probing complex intermolecular interactions and for determining the spatial extension and geometrical characteristics of multicomponent structures composed of diverse molecular constituents, such as proteins, lipids, carbohydrates, nucleic acids, and even cells with viruses. The benefits of fluorescence resonance energy transfer are becoming increasingly evident to researchers who require measurements with high sensitivity, specificity, non-invasiveness, rapidity, and relative simplicity.
Gadella TWJ Jr, Jovin TM, Clegg RM.
Fluorescence lifetime imaging microscopy (FLIM): spatial resolution of microstructures on the nanosecond time scale.
Biophys Chem. 1993; 48(2): 221-239.A frequency domain fluorescence lifetime imaging microscope (FLIM) has been developed. A continuous wave laser excitation source of an epi-illumination fluorescence microscope is modulated at a high frequency fA. The lifetime of the modulated fluorescence emission is determined from the phase delay and modulation depth of the fluorescence signal relative to that of the excitation light. Phase detection is accomplished simultaneously at every location in the image by modulating the high voltage amplification stage of a microchannel plate image intensifier at a frequency near (heterodyne method) or at (homodyne method) fA. The heterodyne or homodyne image output of the intensifier is focused onto a cooled high resolution charge-coupled-device camera for digital recording and subsequent analysis of phase and modulation. The technique has the sensitivity of normal steady state microscopy, and is relatively simple to employ. We present several examples illustrating the applications of FLIM for determining prompt fluorescence lifetimes in ... [truncated at 150 words]
Vámosi G, Gohlke C, Murchie AIH, Lilley DMJ, Clegg RM.
Probing the conformation of DNA structures - 4-way junctions and bulges - with fluorescence.
11th International Biophysics Congress and IUPAB General Assembly at Budapest, Hungary, July 25-30,1993.
Fluorescence studies, especially fluorescence resonance energy transfer (FRET), have aided in determining conformations of complex molecular structures of nucleic acids. Oligonucleotides are synthesized, covalently labeled with dyes, and assembled to form specific structures. For instance, we have applied sensitive fluorescence methods to probe the helical structure of simple specifically labeled DNA duplexes and to investigate more complex structures, such as four-way DNA junctions (a model in solution, of the Holliday genetic recombination junction) and bulged DNA molecules (duplex structures with extra nucleotides in one of the strands). FRET, fluorescence anisotropy and intensity determinations, and kinetic measurements of helix-coil transitions provide us with molecular scale information regarding the conformations of these structures, and the conformational changes ensuing upon perturbations of the molecular environment. Examples will be given and the techniques will be discussed.
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The structure of the four-way junction in DNA.
Annu Rev Biophys Biomol Struct. 1993; 22: 299-328.FOUR-WAY JUNCTIONS AND RECOMBINATION: Branched DNA species are commonly postulated as intermediates in DNA rearrangements. The four-way (Holliday) junction was proposed to be the central intermediate in homologous genetic recombination (6, 39, 81, 88, 89, 92, 93) (Figure 1), and there is good evidence for a role in the integrase class of site-specific recombination events (38, 44, 53, 80). Branched DNA can also arise in other ways, including the replication of DNA as exemplified by bacteriophage T4 replication. In each case, enzymes must interact specifically with these structures to bring about a resolution or repair event. These proteins recognize their DNA substrates at the level of tertiary structure-challenging us to understand how this may be achieved. Despite the potential importance of DNA junctions, their structure has remained poorly understood until relatively recently. The following review describes our current view of the structure of the four-way DNA junction, and the way ... [truncated at 150 words]
Villas-Boas M, Silva JL, Clegg RM.
Pressure studies on protein-DNA interaction.
NATO Advanced Study Institute. Aquafredda di Maratea, Italy. September 20-October 3, 1992.
High Pressure Chemistry, Biochemistry and Materials Sciences (NATO Science Series C, Vol. 401). By R Winter and J Jonas (Editors). Kluwer Academic Press, pp. 579-602, 1993. ISBN: 9780792322900The use of hydrostatic pressure as a tool to study protein-DNA complexes is reported here for two systems: Arc repressor-DNA and nucleosomes. In the former case, it is shown that the magnitude of stabilization of the subunit interaction was correlated with the specificity of the protein-DNA interaction. Pressure-dissociation studies of Arc repressor in the presence of several synthetic DNAs of the same size (24 base pairs) were performed and the largest free energy stabilization of the subunit association was found for the operator DNA sequence. These results demonstrate the importance of free-energy linkage for the protein-DNA recognition process. In a second case, we examine the high pressure perturbation of the intact reconstituted nucleosome structure. The single SH group of H3 histones was labeled specifically with the polar sensitive fluorophore acrylodan and the spectral shift of the emission fluorescence was observed at different pressures and ionic strengths. The emission spectrum shifts ... [truncated at 150 words]
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The structure of branched DNA species.
Q Rev Biophys. 1993; 26(2): 131-175.Branched DNA molecules provide a challenging set of structural problems. Operationally we define branched DNA species as molecules in which double helical segments are interrupted by abrupt discontinuities, and we draw together a number of different kinds of structure in the class, including helical junctions of different orders, and base bulges.
Clegg RM, Murchie AIH, Lilley DMJ.
The four-way DNA junction: a fluorescence resonance energy transfer study.
Braz J Med Biol Res. 1993; 26(4): 405-16.Fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions have been carried out in order to analyze the global structure and its dependence on the concentration of several types of ions. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The results indicate that the four-way junction isomerizes from an unstacked extended square arrangement of the four duplex arms at low ion concentration to an antiparallel stacked X-structure as the salt is added. The ion-related conformational change progresses in a continuous non-cooperative manner as the ionic strength of the solution increases.
Clegg RM, Murchie AIH, Zechel A, Lilley DMJ.
Observing the helical geometry of double-stranded DNA in solution by fluorescence resonance energy transfer.
Proc Natl Acad Sci USA. 1993; 90(7): 2994-8. PMCID: PMC46223The efficiency of fluorescence resonance energy transfer (FRET) between fluorescein and rhodamine covalently attached to both 5' termini of a series of double-stranded DNA species (ranging from 8 to 20 bp) was measured. FRET efficiency varied with a dependence compatible with dye-to-dye distances (R) calculated on the basis of double-stranded B-DNA structure; the helical geometry of double-stranded DNA in solution is clearly evident. The experimental data were consistent with a 1/[1 + (R/R0)6] dependence of FRET efficiency characteristic for the Förster dipole-dipole mechanism. The thermal dissociation of the strands of the duplex DNA species can be followed by using FRET, and from these data we have been able to obtain enthalpies of duplex formation in good agreement with earlier measurements using alternative techniques. FRET measurements at very different salt concentrations can be accurately compared. We conclude that FRET is a reliable and valuable method for studying structure and conformational transitions ... [truncated at 150 words]
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Fluorescence resonance energy transfer and nucleic acids.
DNA Structures Part A: Synthesis and Physical Analysis of DNA (Methods in Enzymology, Vol. 211). Academic Press, pp. 353-388, 1992. ISBN: 0121821129Introduction: Fluorescence resonance energy transfer (FRET) is a spectroscopic process by which energy is passed nonradiatively between molecules over long distances (10- 100 Å). The "donor" molecule (D), which must be a fluorolahore, absorbs a photon and transfer this energy nonradiatively to the "acceptor" molecule (A)...
Duckett DR, Murchie AIH, Bhattacharyya A, Clegg RM, Diekmann S, von Kitzing E, Lilley DMJ.
The structure of DNA junctions and their interaction with enzymes.
Eur J Biochem. 1992; 207(1): 285-95.Branched DNA species are commonly postulated as intermediates in DNA rearrangements, such as genetic recombination. The four-way junction was proposed to be the central intermediate in homologous genetic recombination [1 - 71] and there is good evidence for a role in the integrase class of site-specific recombination events [8 - 11]. Branched DNA can also arise during replication of DNA, such as the replication of T4 DNA. In each case, enzymes are required to interact specifically with these structures, in order to bring about a resolution or repair event. Such proteins achieve impressive feats of molecular recognition at the level of DNA tertiary structure, providing us with a fascinating challenge to understand how this is brought about. However, it has been our belief that the key to this is first to analyze the structure of the DNA substrate, the four-way DNA junction. What follows is a description of our current ... [truncated at 150 words]
Clegg RM, Murchie AIH, Zechel A, Carlberg C, Diekmann S, Lilley DMJ.
Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction.
Biochemistry. 1992; 31(20): 4846-56.We have carried out fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions in order to analyze the global structure and its dependence on the concentration of several types of ions. A knowledge of the structure and its sensitivity to the solution environment is important for a full understanding of recombination events in DNA. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The conclusions are based upon a comparison between a series of many identical DNA molecules which have been labeled on different positions, rather than a determination of a few absolute distances. Details of the FRET analysis are presented; features of the analysis with particular relevance to DNA ... [truncated at 150 words]
Clegg RM, Feddersen BA, Gratton E, Jovin TM.
Time-resolved imaging fluorescence microscopy.
SPIE Conference, Los Angeles, California, 1990.
Time-Resolved Laser Spectroscopy in Biochemistry III (Proceedings of SPIE, Vol. 1640). By JR Lakowicz (Editors). pp. 448-460, 1992. The extension of microscope luminescence measurements into the temporal domain provides the possibility of determining time-resolved properties of microscope samples and their surrounding environments, and thereby extends the conventional steady state measurements. 'Time resolved imaging microscopy' is a relatively new technique whereby fast kinetic and luminescence decay parameters (decay times and the corresponding time or phase resolved amplitudes) are directly and simultaneously measured throughout an image, pixel by pixel, in an optical microscope. Molecular rotation, solvent and matrix relaxation, quenching mechanisms, reactions, and energy transfer are examples of molecular spectroscopic processes that can be studied best by directly measuring the time dependent properties. Dynamic measurements are generally much more informative than their steady state counterparts. The goal of our work is to develop time-resolved methods that can be applied conveniently and routinely to biological material in the microscope over a wide time domain. In addition to the augmented purely spectroscopic ... [truncated at 150 words]
Clegg RM, Marriott GM, Federson BA, Gratton E, Arndt-Jovin DJ, Jovin TM.
Time resolved image microscopy.
36th Annual Meeting of the Biophysical Society, Houston, Texas, 9-13 February 1992.
Biophys J. 1992; 61(2 Pt 2): A415, 2393."Time Resolved Image Microscopy" integrates temporally resolved dynamic properties with the spatial resolution of the light microscope. Fast kinetic and luminescence decay parameters are measured simultaneously at every pixel of a CCD image. We are developing time resolved methods that can be applied conveniently and routinely to biological material in the microscope over a large time domain. In addition to the augmented purely spectroscopic and reaction kinetic information, simultaneous spatial and temporal resolution of an image in a microscope provides significant improvement in image contrast, probe identification and differentiation. For instance, the ability to separate phosphorescence and prompt fluorescence furnishes a new parameter, the ratio of delayed to prompt luminescence at every pixel of the picture, emphasizing particular objects. The time resolution makes it possible to recover structures in an image that are concealed by faster decaying intense luminescence. Examples of these procedures for both delayed luminescence and prompt fluorescence ... [truncated at 150 words]
Feddersen BA, Gratton E, Clegg RM.
Binary solvent studies of Hoechst 33258.
36th Annual Meeting of the Biophysical Society, Houston, Texas, 9-13 February 1992.
Biophys J. 1992; 61(2 Pt 2): A180, 1030.The dye Hoechst 33258 (a bisbenzimidazole derivative) becomes brightly fluorescent when bound to A-T rich sequences of double-stranded DNA. It is useful to stain chromosomal DNA for observation in a microscope; due to the virtual absence of fluorescence interference from dye bound to other cell components or from free dye, the chromosomes are very distinct. To better understand this behavior we have conducted both steady-state and time-resolved fluorescence measurements of Hoechst 33258 in binary solvent mixtures. We have found that the quantum yield of the dye is highly dependent on specific solvent interactions, and not on the bulk properties of the solvent. The time-resolved fluorescence data shows that Hoechst in solution has at least two species. One specie has a fast lifetime (<1 nsec) and a low quantum yield, the other specie has a slower lifetime (-3 nsec) and a much higher quantum yield. The long lifetime component is favored ... [truncated at 150 words]
Marriott GM, Clegg RM, Arndt-Jovin DJ, Jovin TM.
Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.
Biophys J. 1991; 60(6): 1374-87. PMCID: PMC1260198An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a ... [truncated at 150 words]
Clegg RM, Arndt-Jovin DJ.
Spectroscopic analysis of AO bound to RNA and DNA.
XV Congress of the International Society for Analytical Cytology Bergen, Norway, August 25-30, 1991.
Cytometry. 1991; 12(Suppl 5): 103, 549C.We have determined the fluorescence and phosphorescence lifetimes of A0 and its complexes in solution and in the microscope at room temperature, both free and bound to DNA and RNA. A strong green fluorescence (520 nm) with a lifetime of 1.7-1.9 nsec is observed under conditions which are suitable for monomer A0 species. Red fluorescence appears whenever conditions are favorable for dye aggregation. The value of the lifetime varies between 5 and 20 nsec. Phosphorescence of A0 is normally not observed in room temperature solutions because (a) the phosphorescence quantum yield is very low relative to the fluorescence and (b) the oxygen in aqueous solutions is sufficient to quench completely the delayed luminescence. The phosphorescence for natural DNA and RNA in anoxic solutions is observed only in the red and has a lifetime about 1 msec. Our results are contrary to Darzynkiewicz and Kapuscinski, in Flow Cytometry and Sorting, 1990 ... [truncated at 150 words]
Feddersen BA, Gratton E, Clegg RM, Jovin TM.
An optical and electronic heterodyning technique for use with CCD cameras and array detectors for time-resolved fluorescence with sub-nanosecond resolution.
35th Annual Meeting of the Biophysical Society, San Francisco, California, February 1991.
Biophys J. 1991; 59(2 Pt 2): 156a, MI-Po405.Area detectors such as diode arrays and CCD cameras have been employed in frequency domain fluorometry with sub nanosecond resolution. The crucial component is a modulated image intensifier in front of the detector which down converts the high frequencies to a lower frequency which can be handled by the array detector. However, some array devices have a very slow reading frame, on the order of seconds or longer. A single step frequency conversion to the very low frequencies (DC to 1Hz) needed by these detectors is either very expensive or impossible. We have developed a method which uses a two-step frequency conversion to avoid this problem. In the first step the modulation capabilities of the intensifier are used to convert from high frequency (generally 10-100MHz) to an intermediate frequency on the order of 10-500Hz, just as before. This step is done electronically. A second conversion step translates the intermediate frequency ... [truncated at 150 words]
Loontiens FG, McLaughlin LW, Diekmann S, Clegg RM.
Binding of Hoechst 33258 and 4',6'-diamidino-2-phenylindole to self-complementary decadeoxynucleotides with modified exocyclic base substituents.
Biochemistry. 1991; 30(1): 182-9.Fluorescence titrations have been carried out to determine the association constants (Ka) for binding of the dyes Hoechst 33258 and DAPI to the self-complementary decamer d(CTGAATTCAG) and nine duplex derivatives with exocyclic substituent changes in the six central base pairs. Many Ka values are in the range (2-5) x 10(8) (duplex M)-1 at 5.5 degrees C. Replacement of the leftmost adenine by 2-aminopurine in the sequence decreases Ka for Hoechst 33258 by a factor of 170. When the centermost adenine is replaced by 2-aminopurine, Ka for Hoechst 33258 and DAPI is too small to be evaluated. When the centermost adenine is replaced by purine, Ka for both dyes increases, but this very stable duplex-Hoechst 33258 complex is nonfluorescent. The measured affinities are compared to expectations derived from X-ray studies with dodecamer-dye complexes having an identical central binding sequence (Pjura et al., 1987; Teng et al., 1988; Larsen et al., 1989).
Murchie AIH, Clegg RM, von Kitzing E, Duckett DR, Diekmann S, Lilley DMJ.
Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules.
Nature. 1989; 341(6244): 763-6.The four-way junction between DNA helices is the central intermediate in recombination, and the manner of its interaction with resolvase enzymes can determine the genetic outcome of the process. A knowledge of its structure is a prerequisite to understanding the interaction with proteins, and there has been recent progress. Here we use fluorescence energy transfer to determine the relative distances between the ends of a small DNA junction, and hence the path of the strands. Our results are consistent with the geometry of an 'X'. The interconnected helices are juxtaposed so that the continuous strands of each helix generate an antiparallel alignment, and the two interchanged strands do not cross at the centre. The acute angle of the X structure is defined by a right-handed rotation of the helical axes about the axis perpendicular to the X plane, as viewed from the centre of the X.
Piston DW, Marriott GM, Radivoyevich T, Clegg RM, Jovin TM, Gratton E.
Wide-band acousto-optic light modulator for frequency domain fluorometry and phosphorimetry.
Rev Sci Instrum. 1989; 60(8): 2596-2600.Multifrequency-phase and modulation fluorometry allows for accurate analysis of fluorescence decay in the frequency domain. Essential to these frequency domain methods is a high-frequency modulation of the light source. Techniques for generating wide-band modulation of light are currently limited to the use of Pockel's cells and intrinsically modulated sources such as mode-locked lasers and synchrotron radiation. We present a method that employs two acousto-optic modulators in series for use with cw light sources. This modulator system gives two orders of magnitude more intensity output than the Pockel's cell modulator and requires less than one-tenth of the rf driving power. In addition, the Pockel's cell system is limited to modulation frequencies less than 250 MHz, whereas the particular implementation discussed here gives a quasicontinuous distribution of modulation frequencies from dc to 320 MHz. To obtain this range of frequencies, acoustic standing waves are set up simultaneously in each modulator, and the ... [truncated at 150 words]
Jovin TM, Arndt-Jovin DJ, Robert-Nicauld M, Schormann T, Marriott GM, Clegg RM.
Luminescence digital imaging microscopy.
33rd Annual Meeting of the Biophysical Society, Cincinnati, Ohio 1989.
Biophys J. 1989; 55(2 Pt 2): 432a, W-PM-Sym-2.We have applied optical microscopy based on light emission (prompt and delayed fluorescence, phosphorescence) in studies of cellular structures and of processes during the cell cycle, differentiation, and development. Two new technologies have been used [1]: (i) a high performance scientific CCD camera system, and (ii) a confocal laser scanning system. A new method (photobleaching FRET-Digital Imaging Microcopy, pbFRET-DIM) has been developed for spatially resolved proximity measurements by exploiting the high dynamic range, sensitivity, and linearity of the CCD camera in the determination of resonance energy transfer (FRET). Only images of the donor emission before and during photobleaching are required to calculate energy transfer images. Data have been acquired for lectin binding sites, cell surface components involved in exocytosis [2], and for chromatin. The CCD camera has also been adapted for the measurement of delayed luminescence (fluorescence, phosphorescence) by incorporating choppers in the excitation and emission paths of the microscope. ... [truncated at 150 words]
Jovin TM, Marriott GM, Clegg RM, Arndt-Jovin DJ.
Photophysical processes exploited in digital imaging microscopy: fluorescence resonance energy transfer and delayed luminescence.
Ber Bunsen Phys Chem. 1989; 93(3): 387-391.The spectroscopic techniques of fluorescence resonance energy transfer and of time-resolved delayed fluorescence and phosphorescence have been introduced into a microscope equipped with a solid-state CCD camera and phase-locked excitation and emission choppers. The distribution and replication of DNA in cells has been quantitated by these methods, as well as by confocal laser scanning microscopy. Sites of replication in living cells can be uniquely identified by delayed luminescence.
MacGregor RB Jr, Clegg RM, Jovin TM.
Pressure-jump study of the kinetics of ethidium bromide binding to DNA.
Biochemistry. 1985; 24(20): 5503-10.Pressure-jump chemical relaxation has been used to investigate the kinetics of ethidium bromide binding to the synthetic double-stranded polymers poly[d(G-C)] and poly[d(A-T)] in 0.1 M NaCl, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, and 1 mM ethylenediaminetetraacetic acid, pH 7.2, at 24 degrees C. The progress of the reaction was followed by monitoring the fluorescence of the intercalated ethidium at wavelengths greater than 610 nm upon excitation at 545 nm. The concentration of DNA was varied from 1 to 45 microM and the ethidium bromide concentration from 0.5 to 25 microM. The data for both polymers were consistent with a single-step bimolecular association of ethidium bromide with a DNA binding site. The necessity of a proper definition of the ethidium bromide binding site is discussed: it is shown that an account of the statistically excluded binding phenomenon must be included in any adequate representation of the kinetic data. For poly[d(A-T)], the bimolecular association ... [truncated at 150 words]
Loontiens FG, de Boeck H, Clegg RM.
Physicochemical aspects of carbohydrate binding to some plant lectins with binding preference for N-acetylgalactosamine and galactose.
J Biosci. 1985; 8(1-2): 425-436.This contribution illustrates the advantages of some chromophoric and fluorophoric carbohydrate derivatives such asp-nitrophenyl (pNO2Phe) or 4-methylumbelliferyl (MeUmb) glycosides andN-dansylgalactosamine in studies of the binding equilibrium and kinetics with some plant lectins. The methods used involve continuous titrations of changes in ligand or protein absorption and ligand fluorescence, including substitution titrations as well as stopped-flow, temperature-jump or pressure-jump relaxation kinetics.
When monitored by temperature-jump relaxation, binding of MeUmbαGal to the bloodgroup A specific lectin GSAI-A4 fromGriffonia simplicifolia is a simple bimolecular association with parametersk + = 9.4 × 104 M-1 s-1 andk -1 = 5.3 s-1 at 23°C, but binding to the GSAI-B4 lectin is biphasic.
The complementarity of the peanut agglutinin binding site with Galβ1 → 3GalNAc that occurs in manyO-glycoproteins follows from enthalpic considerations and also from the value of the dissociation-rate parameterk -1 = 0.24 s-1 of the MeUmbβGalβl → 3GalNAc.lectin complex. This value, obtained by stopped-flow kinetics is ... [truncated at 150 words]
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Translational diffusion of proteins and lipids in artificial lipid bilayer membranes. A comparison of experiment with theory.
Progress in Protein-Lipid Interactions (Progress in Protein-Lipid Interactions, Vol. 1). By A Watts (Editors). Elsevier Science Ltd, pp. 173-229, 1985. ISBN: 9780444806307
de Boeck H, MacGregor RB Jr, Clegg RM, Sharon N, Loontiens FG.
Binding of N-dansylgalactosamine to the lectin from Erythrina cristagalli as followed by stopped-flow and pressure-jump relaxation kinetics.
Eur J Biochem. 1985; 149(1): 141-5.The binding kinetics of N-dansylgalactosamine to the lectin from Erythrina cristagalli have been studied using stopped-flow and pressure-jump chemical relaxation by monitoring ligand fluorescence. Both methods gave results which are consistent with a simple bimolecular association reaction. The association rate constant, k+1 = 4.8 × 10(4) M-1 s-1, is far too low to be controlled by diffusion; the dissociation rate is 0.4-0.66 s-1, depending upon the method of determination and the experimental conditions. Identical reaction-rate parameters were obtained at pH 7.3, where soluble aggregates can be present in the lectin solution and at pH 4.7 where such aggregates are absent. The slow rates of carbohydrate binding seem to be characteristic for most lectins and lend support to the idea that they are evolutionary related and have structurally similar binding sites. Analysis of the relaxation amplitudes of the pressure-jump experiments yielded a molar reaction volume change, delta V0, upon binding of ... [truncated at 150 words]
Vaz WL, Hallmann D, Clegg RM, Gambacorta A, de Rosa M.
A comparison of the translational diffusion of a normal and a membrane-spanning lipid in L alpha phase 1-palmitoyl-2-oleoylphosphatidylcholine bilayers.
Eur Biophys J. 1985; 12(1): 19-24.We have used the fluorescence recovery after photobleaching technique to study the translational diffusion, in L alpha phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), of fluorescent derivatives of 1-palmitoyl-2-oleoylphosphatidylethanolamine (NBD-POPE) and a membrane-spanning phosphatidylethanolamine (NBD-MSPE). The latter derivative was prepared from a membrane-spanning glycerol-dialkyl-glycerol tetraether lipid isolated from the thermophilic and acidophilic archaebacterium Sulfolobus solfataricus. The translational diffusion was examined between about 15 degrees and 45 degrees C. It is shown that over this temperature range the translational diffusion coefficient for NBD-MSPE is approximately 2/3 that for NBD-POPE which spans only one monolayer of the bilayer. The result is interpreted in terms of existing models for translational diffusion in lipid membranes.
Regenfuss P, Clegg RM, Fulwyler MJ, Barrantes FJ, Jovin TM.
Mixing liquids in microseconds.
Rev Sci Instrum. 1985; 56(2): 283-290.An instrument is described in which two solutions can be homogeneously mixed within several microseconds. The liquids flow separately through two coaxial capillaries with conical tips and then simultaneously around a sphere (50–100 µ in diameter) which has been positioned close to the end of the outer tip. The liquids flow with velocities of ~100 m/s through the small passages (~5 µ wide) separating the sphere and the wall of the outer capillary and mix in the turbulent liquid flow behind the sphere. The mixed liquids are then ejected as a narrow liquid jet for observation. Design characteristics and construction techniques are presented along with a discussion of the properties of the turbulent flow field and estimates of the expected practically realizable mixing times. The experimentally determined speed of mixing indicates that we have nearly achieved the proposed lower limits of the mixing time.
Vaz WL, Clegg RM, Hallmann D.
Translational diffusion of lipids in liquid crystalline phase phosphatidylcholine multibilayers. A comparison of experiment with theory.
Biochemistry. 1985; 24(3): 781-6.A systematic study of the translational diffusion of the phospholipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) has been undertaken in liquid crystalline phase phosphatidylcholine bilayers by using the fluorescence recovery after photobleaching technique. This work was done with the intention of comparing the experimental results with the predictions of theoretical models for diffusion in membranes. The following is shown. For NBD-PE, the dependence of the translational diffusion coefficient (Dt) upon the acyl chain length of the diffusant is not that predicted by continuum fluid hydrodynamic models for diffusion in membranes [Saffman, P.G., & Delbrueck, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3111-3113; Hughes, B. D., Pailthorpe, B. A., & White, L. R. (1981) J. Fluid Mech. 110, 349-372]. Plots of Dt vs. 1/T (Arrhenius plots) are nonlinear in dilauroyl-phosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayers where the acyl chain composition of the NBD-PE is matched with that of the ... [truncated at 150 words]
van Landschoot A, Loontiens FG, Clegg RM, Sharon N, de Bruyne CK.
Binding of 4-methylumbelliferyl n-acetyl-chitooligosaccharides to wheat-germ agglutinin. A reinvestigation of equilibrium studies.
Eur J Biochem. 1977; 79(1): 275-83.The binding of 4-methylumbelliferyl per-N-acetyl-chitooligosaccharides, MeUmbGlcNAc (I), MeUmb(GlcNAc)2 (II) and MeUmb(GlcNAc)3 (III) to wheat germ agglutinin, was studied by equilibrium dialysis, difference absorption spectrometry and extrinsic fluorescence quenching [Privat, J. P., Delmotte, F. & Monsigny, M. (1974) FEBS Lett. 46, 229–232] titration at a fixed wavelength or above 370 nm. The change in optical properties of the MeUmb group upon binding of the glycosides to the agglutinin depends on the length of the carbohydrate chain. An intense and carbohydrate-specific difference absorption spectrum was observed with I (-Δe at 316 nm equal to 2770 M-1 cm-1), a weaker one with II (-Δe at 316 nm = 1070 M-1 cm-1), but none was observed with III; the fluorescence quenching is 100% for compounds I and II and 44% for compound III. As determined with I, using equilibrium dialysis and difference absorption spectrometry, there are two identical and independent binding sites per subunit. ... [truncated at 150 words]
Loontiens FG, Clegg RM, van Landschoot A, Jovin TM.
Binding of 4-methylumbelliferyl alpha-D-mannopyranoside to tetrameric Concanavalin A Fluorescence temperature-jump relaxation study.
Eur J Biochem. 1977; 78(2): 465-9.The kinetics of saccharide binding to the treatment form of concanavalin A have been studies at pH 7.2 with the temperature-jump method. 4-Methylumbelliferyl alpha-D-mannopyranoside was used as a ligand; its fluorescence is totally quenched upon binding. A single relaxation of ligand fluorescence (tau = 20-400 ms) was observed and was investigated at three different temperatures, using kinetic titration and dilution types of experiments. The concentration dependence of the relaxation time and amplitude was consistent with a single-step bimolecular association and independent binding sites. In the temperature range 13-24 degrees C the association and dissociation rate parameters are in the range (6-10) × 10(4) M-1 s-1 and (1.4-3.2)s-1 respectively, corresponding to activation energies for the forward and reverse reactions equal to approx. 13 and 8 kcal/mol (54 and 33 kJ/mol) respectively. Two additional relaxations of protein fluorescence (3 ms and larger than 1 s at 25 degrees C) were unaffected by ... [truncated at 150 words]
van Landschoot A, Clegg RM, Loontiens FG, Jovin TM.
Binding of the 4 methylumbelliferyl glycosides of alpha-mannobiose and mannotriose to tetrameric Concanavalin A: equilibrium and fast kinetic studies using fluorescence quenching.
Arch Int Physiol Biochim. 1977; 85(1): 203-4.To further our understanding on the binding of lectins to cellwall receptors, we have undertaken a kinetic study of the binding mechanism of carbohydrates as a function of their chain length. As no change of protein fluorescence occurs upon binding of simple carbohydrates to concanavalin A (con A), we have used 4-methylumbelliferyl a-D-mannobioside and -mannotrioside (MUM2, MUM3). These possess alpha(1->2) linkages and were prepared through their acetochloro derivatives, obtained from the separated acetolysis products of the cell-wall mannan from Saccharomyces cerevisiae.
Loontiens FG, Clegg RM, Jovin TM.
Binding of 4-methylumbelliferyl alpha-d-mannopyranoside to dimeric and tetrameric Concanavalin A: equilibria and kinetic study using temperature-jump relaxation of fluorescence quenching.
Arch Int Physiol Biochim. 1977; 85(1): 184-6.The binding equilibrium of 4-methylumbelliferyl a-D-mannopyranoside (MUM) and fully metallized concanavalin A , composed of intact polypeptide chains (con A), was characterized (LOONTIENS
et al., 1977) by equilibrium dialysis, difference spectroscopy of ligand absorption with minima at 334 and 322 nm and by continuous titration of the total quenching (DEAN & HOMER, 1973) of ligand fluorescence.
Clegg RM, Loontiens FG, Jovin TM.
Binding of 4-methylumbelliferyl alpha-D-mannopyranoside to dimeric Concanavalin A: fluorescence temperature-jump relaxation study.
Biochemistry. 1977; 16(2): 167-75.The kinetics of saccharide binding to the dimer form of concanavalin A (con A) has been studied at pH 5.5 with the fluorescence temperature-jump method. 4-Methylumbelliferyl alpha-D-mannopyranoside, a fluorescent carbohydrate derivative which is quenched upon binding to con A, was used as the ligand. Three relaxation effects were seen. The major relaxation (r = 20-400 ms) was investigated at four different temperatures. The behaviour of this relaxation as a function of reactant concentrations is consistent with a simple one-step bimolecular association reaction. These conclusions result from the analysis of both the relaxation times and amplitudes, and from the comparison of the kinetically determined equilibrium parameters (Kass = 3.5 × 10(4) M-1 at 18.5 degrees C, delta H degrees = -(6-7) kcal/mol) to those obtained from a parallel series of equilibrium experiments (Loontiens, F.G., Clegg R.M., and Jovin, T.M. (1977), Biochemistry 16, preceding paper in this issue). The association and dissociation ... [truncated at 150 words]
Loontiens FG, Clegg RM, Jovin TM.
Binding of 4-methylumbelliferyl alpha-D-mannopyranoside to tetrameric and unmodified or derivatized dimeric Concanavalin A: equilibrium studies.
Biochemistry. 1977; 16(2): 159-66.The binding of 4-methylumbelliferyl alpha-D-mannopyranoside (MUM) and concanavalin A, composed of intact polypeptide chains, was studied by equilibrium dialysis, difference spectroscopy, and fluorescence titration (Dean, B.R., and Homer, R.B. (1973), Biochim. Biophys. Acta. 322, 141-144), measured either at a fixed wavelength or above 350 nm. Dimeric and tetrameric concanavalin A samples were used under conditions of apparently full metal saturation. The results are consistent with a single carbohydrate-specific site per protomer, without interaction between sites; no indication for additional unspecific binding could be obtained. The values of the association constant are independent of the method or of the saturation range used and 4-methylumbelliferyl alpha-D-mannopyranoside, bound at a fractional saturation of 0.91 can be totally displaced by methyl alpha-D-mannopyranoside. The thermodynamic binding parameters for acetylated or succinylated concanavalin A, composed of intact polypeptide chains, were obtained by titration of total MUM fluorescence in the temperature range 9-39 degrees C. For unmodified ... [truncated at 150 words]
