The Laboratory for Fluorescence Dynamics (LFD) should be acknowledged in any publications arising from the data collected in the LFD using the following text:
Fong AH, Romero-López M, Heylman CM, Keating M, Tran D, Sobrino A, Tran AQ, Pham HH, Fimbres C, Gershon PD, Botvinick EL, George SC, Hughes CCW.
Three-Dimensional adult cardiac extracellular matrix promotes maturation of human induced pluripotent stem cell-derived cardiomyocytes.
Tissue Eng Part A. 2016; 22(15-16): 1016-1025. PMCID: PMC4991595Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular, human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency, their self-renewal potential, and their ability to create patient-specific cell lines. Unfortunately, pluripotent stem cell-derived CMs are immature, with characteristics more closely resembling fetal CMs than adult CMs, and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation, as does the geometry of the environment—two-dimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSC-derived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold, compared with 2D culture. 3D cardiac ECM promoted increased expression of calcium-handling genes, Junctin, CaV1.2, NCX1, HCN4, ... [truncated at 150 words]
Steinhardt RC, O’Neill JM, Rathbun CM, McCutcheon DC, Paley MA, Prescher JA.
Design and synthesis of an Alkynyl Luciferin analogue for bioluminescence imaging.
Chem Eur J. 2016; 22(11): 3671-3675.Herein, the synthesis and characterization of an alkyne-modified luciferin is reported. This bioluminescent probe was accessed using C−H activation methodology and was found to be stable in solution and capable of light production with firefly luciferase. The luciferin analogue was also cell permeant and emitted more redshifted light than d-luciferin, the native luciferase substrate. Based on these features, the alkynyl luciferin will be useful for a variety of imaging applications.
Kim MY, Li DJ, Pham LK, Wong BG, Hui EE.
Microfabrication of high-resolution porous membranes for cell culture.
J Memb Sci. 2014; 452: 460-469. PMCID: PMC3931465Microporous membranes are widely utilized in cell biology to study cell–cell signaling and cell migration. However, the thickness and low porosity of commercial track-etched membranes limit the quality of cell imaging and the degree of cell–cell contact that can be achieved on such devices. We employ photolithography-based microfabrication to achieve porous membranes with pore diameter as small as 0.9 µm, up to 40% porosity, and less than 5% variation in pore size. Through the use of a soap release layer, membranes as thin as 1 µm can be achieved. The thin membranes minimally disrupt contrast enhancement optics, thus allowing good quality imaging of unlabeled cells under white light, unlike commercial membranes. In addition, the polymer membrane materials display low autofluorescence even after patterning, facilitating high quality fluorescence microscopy. Finally, confocal imaging suggests that substantial cell–cell contact is possible through the pores of these thin membranes. This membrane technology can enhance ... [truncated at 150 words]
Lu D, Kan JJ, Fullerton EE, Liu Z.
Enhancing spontaneous emission rates of molecules using nanopatterned multilayer hyperbolic metamaterials.
Nat Nanotechnol. 2014; 9(1): 48-53.Plasmonic nanostructures have been extensively used to manipulate the spontaneous light emission rate of molecules and their radiative efficiency. Because molecules near a metallic surface experience a different environment than in free space, their spontaneous radiative emission rate is generally enhanced. Such enhancement, measured by means of the Purcell factor, arises as a consequence of the overlap between the surface plasmon mode frequency and the emission spectrum of the molecule. However, such overlap is available only for a few narrow bands of frequency due to the limited plasmonic materials existing in nature. Although this limitation can be overcome by using hyperbolic metamaterials (HMMs)-a type of nanoscale artificial material with hyperbolic dispersion relations-the Purcell factor and the radiative power have remained relatively low. Here, we show that by nanopatterning a hyperbolic metamaterial made of Ag and Si multilayers, the spontaneous emission rate of rhodamine dye molecules is enhanced 76-fold at tunable ... [truncated at 150 words]
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Understanding the intracellular dynamics of endogenous ribonucleic acid species in living cells.
PhD in University of Western Sydney, 2012.
Advisor: Mark Jones
The endogenous properties of RNA are often hard to examine due to fragility of the species upon extraction, labelling difficulties and further, an inability to monitor real time dynamics. Described in this thesis are multiple developed models which enable the real time analysis of transcripts inside living cells. Through the application of fluorescence microscopy, analysis techniques and a small RNA specific probe, the natural properties of endogenous transcripts were visualised.
The fluorophore Pyronin Y (PY) provided a small cationic probe which in a concentration dependent manner may be applied to live cells to enable visualisation of double stranded RNA through the aid of microscopy. The entry mechanisms of the fluorophore were first examined and identified to be dependent upon membrane polarity, with a slight negative charge required for attachment of the probe to the membrane followed by flipping of the probe and release into the matrix. This confirmed that the probe ... [truncated at 150 words]
Lei TC, Glazner GF, Duffy M, Scherrer L, Pendyala S, Lib B, Wang X, Wang H, Huang Z.
Optical properties of hematoporphyrin monomethyl ether (HMME), a PDT photosensitizer.
Photodiagnosis Photodyn Ther. 2012; 9(3): 232-242.We report on some of the optical properties of Hemoporfin (hematoporphyrin monomethyl ether, HMME), a photodynamic therapy (PDT) photosensitizer that has been in clinical trials in China since the early 1990s. We characterized the photosensitizer on the basis of one- and two-photon absorption and fluorescence emission. The effects of photobleaching were probed to characterize its decay kinetics. Additionally, we determined time resolved fluorescence and thermal effects on fluorescence and absorption properties.
McCutcheon DC, Paley MA, Steinhardt RC, Prescher JA.
Expedient synthesis of electronically modified luciferins for bioluminescence imaging.
J Am Chem Soc. 2012; 134(18): 7604-7607. PMCID: PMC3613990Bioluminescence imaging with luciferase enzymes requires access to light-emitting, small-molecule luciferins. Here, we describe a rapid method to synthesize d-luciferin, the substrate for firefly luciferase (Fluc), along with a novel set of electronically modified analogues. Our procedure utilizes a relatively rare, but synthetically useful dithiazolium reagent to generate heteroaromatic scaffolds in a divergent fashion. Two of the luciferin analogues produced with this approach emit light with Fluc in vitro and in live cells. Collectively, our work increases the number of substrates that can be used for bioluminescence imaging and provides a general strategy for synthesizing new collections of luciferins.
Carrion B, Huang CP, Ghajar CM, Kachgal S, Kniazeva E, Jeon NL, Putnam AJ.
Recreating the perivascular niche ex vivo using a microfluidic approach.
Biotechnol Bioeng. 2010; 107(6): 1020-1028. PMCID: PMC3367510Stem cell niches are composed of numerous microenvironmental features, including soluble and insoluble factors, cues from other cells, and the extracellular matrix (ECM), which collectively serve to maintain stem cell quiescence and promote their ability to support tissue homeostasis. A hallmark of many adult stem cell niches is their proximity to the vasculature in vivo, a feature common to neural stem cells, mesenchymal stem cells (MSCs) from bone marrow and adipose tissue, hematopoietic stem cells, and many tumor stem cells. In this study, we describe a novel 3D microfluidic device (MFD) as a model system in which to study the molecular regulation of perivascular stem cell niches. Endothelial cells (ECs) suspended within 3D fibrin gels patterned in the device adjacent to stromal cells (either fibroblasts or bone marrow-derived MSCs) executed a morphogenetic process akin to vasculogenesis, forming a primitive vascular plexus and maturing into a robust capillary network with hollow ... [truncated at 150 words]
Weise K, Triola G, Janosch S, Waldmann H, Winter R.
Visualizing association of lipidated signaling proteins in heterogeneous membranes - Partitioning into subdomains, lipid sorting, interfacial adsorption, and protein association.
Biochim Biophys Acta. 2010; 1798(7): 1409-1417.In a combined chemical biological and biophysical approach, we studied the partitioning of differently fluorescent-labeled palmitoyl and/or farnesyl lipidated peptides, which represent membrane recognition model systems, as well as the full lipidated N-Ras protein into various model membrane systems including canonical model raft mixtures. To this end, two-photon fluorescence microscopy on giant unilamellar vesicles, complemented by tapping-mode atomic force microscopy (AFM) measurements, was carried out. The measurements were performed over a wide temperature range, ranging from 30 to 80 °C to cover different lipid phase states (solid-ordered (gel), fluid/gel, liquid-ordered/liquid-disordered, all-fluid). The results provide direct evidence that partitioning of the lipidated peptides and N-Ras occurs preferentially into liquid-disordered lipid domains, which is also reflected in a faster kinetics of incorporation. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is liquid-disordered > liquid-ordered ≫ solid-ordered. Intriguingly, we detect – using the better spatial resolution of AFM – ... [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]
Kang I, Reem RE, Kaczmarowski AL, Malpeli JG.
Contrast sensitivity of cats and humans in scotopic and mesopic conditions.
J Neurophysiol. 2009; 102(2): 831-840. PMCID: PMC2724355Human contrast sensitivity in low scotopic conditions is regulated according to the deVries–Rose law. Previous cat behavioral data, as well as cat and mice electrophysiological data, have not confirmed this relationship. To resolve this discrepancy at the behavioral level, we compared sensitivity in dim light for cats and humans in parallel experiments using the same visual stimuli and similar behavioral paradigms. Both species had to detect Gabor functions (SD = 1.5°, spatial frequencies from 0 to 4 cpd, temporal frequency 4 Hz) presented 8° to the right or left of a central fixation point over an 8 log-unit range of adaptation levels spanning scotopic vision and extending well into the mesopic range. Cats had 0.74 log unit greater absolute sensitivity than that of humans for spatial frequencies ≤1/8 cpd. Cats had better contrast sensitivity overall for spatial frequencies <1/2 cpd, whereas humans were more sensitive for spatial frequencies above this. ... [truncated at 150 words]
Bailey RW, Nguyen T, Robertson L, Gibbons E, Nelson J, Christensen RE, Bell JP, Judd AM, Bell JD.
Sequence of physical changes to the cell membrane during glucocorticoid-induced apoptosis in S49 lymphoma cells.
Biophys J. 2009; 96(7): 2709-2718. PMCID: PMC2711280During apoptosis, physical changes in the plasma membrane prepare the cell for clearance by phagocytes and hydrolysis by secretory phospholipase A2 (sPLA2). The relationships among these changes have not been adequately established, especially for hormone-stimulated apoptosis. This study addresses these issues for glucocorticoid-induced apoptosis in S49 lymphoma cells. Flow cytometry, microscopy, and fluorescence spectroscopy were used to assess merocyanine 540 emission, laurdan generalized polarization, phosphatidylserine exposure, caspase activation, and membrane permeability to propidium iodide in the absence and presence of sPLA2. The earliest event observed was activation of cellular caspases. Results with membrane probes suggest that interlipid spacing also increases early during apoptosis and precedes transbilayer migration of phosphatidylserine, DNA fragmentation, and a general increase in lipid order associated with blebbing and dissolution of the cells. The activity of sPLA2 appeared to be linked more to lipid spacing than to loss of membrane asymmetry. The early nature of some of ... [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.
Stott BM, Vu MP, McLemore CO, Lund MS, Gibbons E, Brueseke TJ, Wilson-Ashworth HA, Bell JD.
Use of fluorescence to determine the effects of cholesterol on lipid behavior in sphingomyelin liposomes and erythrocyte membranes.
J Lipid Res. 2008; 49(6): 1202-1215. PMCID: PMC2386901The purpose of this study was to generate the equivalent of a cholesterol/temperature phase map for a biological membrane using fluorescence spectroscopy. The pseudo-phase map was created using human erythrocytes treated with various concentrations of methyl-β-cyclodextrin to remove defined amounts of cholesterol and a trio of fluorescent probes that assess different membrane properties (laurdan, diphenylhexatriene, and merocyanine 540). Parallel experiments with two-photon microscopy suggested that changes in cellular cholesterol content affected the entire membrane rather than being localized to specific macroscopic domains. The various regions of the composite erythrocyte pseudo-phase map were interpreted using analogous data acquired from multilamellar vesicles that served as simplified models of cholesterol-dependent phases. The vesicles consisted of various concentrations of cholesterol (0 to 50 mol%) with either palmitoyl sphingomyelin, 1:1 dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine, or phospholipid mixtures intended to simulate either the inner or outer leaflet of erythrocyte membranes. Four distinguishable regions were observed in sphingomyelin ... [truncated at 150 words]
Heiner AL, Gibbons E, Fairbourn JL, Gonzalez LJ, McLemore CO, Brueseke TJ, Judd AM, Bell JD.
Effects of cholesterol on physical properties of human erythrocyte membranes: impact on susceptibility to hydrolysis by secretory phospholipase A2.
Biophys J. 2008; 94(8): 3084-3093. PMCID: PMC2275687The ability of secretory phospholipase A2 (sPLA2) to hydrolyze cell membranes is highly dependent on the physical properties of the membrane. The effects of cholesterol on these properties have been characterized in artificial bilayers and found to alter sPLA2 activity significantly. It is hypothesized that the natural difference in cholesterol content between erythrocytes and leukocytes is in part responsible for their differing susceptibility to hydrolysis by sPLA2. To test this hypothesis, defined amounts of cholesterol were removed from erythrocyte membranes using methyl-β-cyclodextrin. Treatment of cells with methyl-β-cyclodextrin increased the hydrolysis rate and total substrate hydrolyzed by sPLA2. In general, this effect of cholesterol removal was more pronounced at higher temperatures. Comparison of the level of membrane order (assessed with the fluorescent probe laurdan) with hydrolysis rate revealed that sPLA2 activity was greatly enhanced upon significant reductions in lipid order. Additional treatment of the cells with calcium ionophore further enhanced the ... [truncated at 150 words]
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Mechanisms by which apoptotic membranes become susceptible to secretory phospholipase A2.
Master in Physiology and Developmental Biology, Brigham Young University, 2008.
Advisor: John D Bell
During apoptosis, changes occur in T-lymphocyte membranes that render them susceptible to hydrolysis by secretory phospholipase A2 (sPLA2). To study the relevant mechanisms, a simplified model of apoptosis using a calcium ionophore was first applied. Kinetic and flow cytometry experiments provided key observations regarding ionophore treatment: initial hydrolysis rate was elevated, total reaction product was increased four-fold, and adsorption of the enzyme to the membrane surface was unaltered. Analysis of these results suggested that susceptibility during calcium-induced apoptosis is limited by substrate availability rather than enzyme adsorption. Fluorescence experiments identified three membrane alterations that might affect substrate access to the sPLA2 active site. First, intercalation of merocyanine 540 into the membrane was improved, suggesting increased lipid spacing. Second, laurdan detected increased solvation of the lower head group region of the membrane. Third, the rate at which fluorescent lipids could be removed from the membrane by albumin was enhanced, implying greater ... [truncated at 150 words]
Buranachai C, Kamiyama D, Chiba A, Williams BD, Clegg RM.
Rapid frequency-domain FLIM spinning disk confocal microscope: lifetime resolution, image improvement and wavelet analysis.
J Fluoresc. 2008; 18(5): 929-942.A spinning disk confocal attachment is added to a full-field real-time frequency-domain fluorescence lifetime-resolved imaging microscope (FLIM). This provides confocal 3-D imaging while retaining all the characteristics of the normal 2-D FLIM. The spinning disk arrangement allows us to retain the speed of the normal 2-D full field FLIM while gaining true 3-D resolution. We also introduce the use of wavelet image transformations into the FLIM analysis. Wavelets prove useful for selecting objects according to their morphology, denoising and background subtraction. The performance of the instrument and the analysis routines are tested with quantitative physical samples and examples are presented with complex biological samples.
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Multi scale fluorescence studies: the spinning disk confocal fluorescence lifetime imaging microscopy & single molecule nano-metronome & single molecule study of tetramethyrhodamine-DNA interaction.
PhD in Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 2008.
Advisor: Robert M Clegg
The thesis is consisted of three different sections independent of each other. The first part involves the frequency domain fluorescence lifetime imaging microscopy (FLIM) and an improvement of FLIM setup to decrease the out of focus background fluorescence without drastically sacrificing the data acquisition speed while taking fluorescence lifetime data. The setup incorporates a spinning disk confocal unit to the conventional wide-field fluorescence lifetime imaging; therefore, it is a marriage between confocality and data acquisition speed. The detailed discussion about the principle of the setup in conjunction to the benefits when the setup is used for imaging thick specimens is presented. In addition, a new concept of using the Wavelets analysis on fluorescence lifetime images is introduced. Besides facilitating morphology recognition and noise reduction usually shown in normal image analysis, the benefit of wavelets analysis on decomposing an image into different spatial frequencies proves to be useful in removing the ... [truncated at 150 words]
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Sucrose synthase oligomerization and F-actin association are regulated by sucrose concentration and phosphorylation.
Plant Cell Physiol. 2007; 48(11): 1612-23.Sucrose synthase (SUS) is a key enzyme in plant metabolism, as it serves to cleave the photosynthetic end-product sucrose into UDP-glucose and fructose. SUS is generally assumed to be a tetrameric protein, but results in the present study suggest that SUS can form dimers as well as tetramers and that sucrose may be a regulatory factor for the oligomerization status of SUS. The oligomerization of SUS may also affect the cellular localization of the protein. We show that sucrose concentration modulates the ability of SUS1 to associate with F-actin in vitro and that calcium-dependent protein kinase-mediated phosphorylation of recombinant SUS1 at the Ser15 site is a negative regulator of its association with actin. Although high sucrose concentrations and hyperphosphorylation have been shown to promote SUS association with the plasma membrane, we show that the opposite is true for the SUS-actin association. We also show that SUS1 has a unique 28 ... [truncated at 150 words]
Bailey RW, Olson ED, Vu MP, Brueseke TJ, Robertson L, Christensen RE, Parker KH, Judd AM, Bell JD.
Relationship between membrane physical properties and secretory phospholipase A2 hydrolysis kinetics in S49 cells during ionophore-induced apoptosis.
Biophys J. 2007; 93(7): 2350-62. PMCID: PMC1965435During apoptosis, changes occur in lymphocyte membranes that render them susceptible to hydrolysis by secretory phospholipase A(2) (sPLA(2)). To study the relevant mechanisms, a simplified model of apoptosis using a calcium ionophore was applied. Kinetic and flow cytometry experiments provided key observations regarding ionophore treatment: the initial rate of hydrolysis was elevated at all enzyme concentrations, the total amount of reaction product was increased fourfold, and adsorption of the enzyme to the membrane surface was unaltered. Analysis of these results suggested that susceptibility during calcium-induced apoptosis is limited by availability of substrate rather than adsorption of enzyme. Fluorescence experiments identified three membrane alterations during apoptosis that might affect substrate access to the sPLA(2) active site. First, intercalation of merocyanine 540 into the membrane was improved, suggesting an increase in lipid spacing. Second, laurdan detected increased solvation of the lower headgroup region of the membrane. Third, the rate at which fluorescent ... [truncated at 150 words]
Guest CB, Hartman ME, O'Connor JC, Chakour KS, Sovari AA, Freund GG.
Phagocytosis of cholesteryl ester is amplified in diabetic mouse macrophages and is largely mediated by CD36 and SR-A.
PLoS One. 2007; 2(6): e511. PMCID: PMC1876806Type 2 diabetes (T2D) is associated with accelerated atherosclerosis, which accounts for approximately 75% of all diabetes-related deaths. Here we investigate the link between diabetes and macrophage cholesteryl ester accumulation. When diabetic (db/db) mice are given cholesteryl ester intraperitoneally (IP), peritoneal macrophages (PerMΦs) recovered from these animals showed a 58% increase in intracellular cholesteryl ester accumulation over PerMΦs from heterozygote control (db/+) mice. Notably, PerMΦ fluid-phase endocytosis and large particle phagocytosis was equivalent in db/+and db/db mice. However, IP administration of CD36 and SR-A blocking antibodies led to 37% and 25% reductions in cholesteryl ester accumulation in PerMΦ. Finally, in order to determine if these scavenger receptors (SRs) were part of the mechanism responsible for the increased accumulation of cholesteryl esters observed in the diabetic mouse macrophages, receptor expression was quantified by flow cytometry. Importantly, db/db PerMΦs showed a 43% increase in CD36 expression and an 80% increase in SR-A ... [truncated at 150 words]
Cuevas FJ, Jameson DM, Sotomayor CP.
Modulation of reconstituted pig kidney Na+/K(+)-ATPase activity by cholesterol in endogenous lipid vesicles: role of lipid domains.
Biochemistry. 2006; 45(46): 13855-68.Diverse experimental and theoretical evidence suggests that plasma membranes contain cholesterol-induced segregated domains that could play a key role in the modulation of membrane functions, including intrinsic enzyme activity. To gain insight into the role of cholesterol, we reconstituted pig kidney Na+/K+-ATPase into unilamellar vesicles of endogenous lipids mimicking the natural membrane and addressed the question of how modification of the cholesterol content could affect the ATPase activity via changes in the membrane lipid phase and in the protein structure and dynamics. We used steady-state and time-resolved fluorescence spectroscopy with the lipid phase probes DPH and Laurdan and the protein probe fluorescein and also used infrared spectroscopy using attenuated total reflectance. Upon modification of membrane cholesterol content, the ATPase activity did not change monotonically but instead exhibited abrupt changes resulting in two peaks at or close to critical cholesterol mole fractions (25 and 33.3 mol %) predicted by the superlattice ... [truncated at 150 words]
Fidorra M, Duelund L, Leidy C, Simonsen AC, Bagatolli LA.
Absence of fluid-ordered/fluid-disordered phase coexistence in ceramide/POPC mixtures containing cholesterol.
Biophys J. 2006; 90(12): 4437-51. PMCID: PMC1471871The effect of temperature on the lateral structure of lipid bilayers composed of porcine brain ceramide and 1-palmitoyl 2-oleoyl-phosphatidylcholine (POPC), with and without addition of cholesterol, were studied using differential scanning calorimetry, Fourier transformed infrared spectroscopy, atomic force microscopy, and confocal/two-photon excitation fluorescence microscopy (which included LAURDAN generalized polarization function images). A broad gel/fluid phase coexistence temperature regime, characterized by the presence of micrometer-sized gel-phase domains with stripe and flowerlike shapes, was observed for different POPC/ceramide mixtures (up to approximately 25 mol % ceramide). This observed phase coexistence scenario is in contrast to that reported previously for this mixture, where absence of gel/fluid phase coexistence was claimed using bulk LAURDAN generalized polarization (GP) measurements. We demonstrate that this apparent discrepancy (based on the direct comparison between the LAURDAN GP data obtained in the microscope and the fluorometer) disappears when the additive property of the LAURDAN GP function is taken into ... [truncated at 150 words]
Breusegem SY, Levi M, Barry NP.
Fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy.
Nephron Exp Nephrol. 2006; 103(2): e41-e49.With few and commercially available add-ons, both confocal and full-field fluorescence microscopes can be adapted to provide more information on the biological sample of interest. In this review we discuss the possibilities offered by two additional functionalities to fluorescence microscopes, fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging mi croscopy (FLIM). FCS measurements at a single point in a sample allow kinetic and diffusion properties of fluorescently labeled molecules to be determined, as well as their concentration and aggregation state. Data from multiple points of the sample can be acquired using scanning-FCS, image correlation spectroscopy, and raster image correlation spectroscopy. These techniques cover phenomena with characteristic durations from sub-microsecond to second time scales. The power of FLIM lies in the fact that the measured fluorescent lifetime of a fluorophore is sensitive to the molecular environment of that fluorophore. FLIM is a robust means to quantify Förster resonance energy transfer and ... [truncated at 150 words]
Jeng ES, Moll AE, Roy AC, Gastala JB, Strano MS.
Detection of DNA hybridization using the near-infrared band-gap fluorescence of single-walled carbon nanotubes.
Nano Lett. 2006; 6(3): 371-375.We demonstrate the optical detection of DNA hybridization on the surface of solution suspended single-walled carbon nanotubes (SWNTs) through a SWNT band gap fluorescence modulation. Hybridization of a 24-mer oligonucleotide sequence with its complement produces a hypsochromic shift of 2 meV, with a detection sensitivity of 6 nM. The energy shift is modeled by correlating the surface coverage of DNA on SWNT to the exciton binding energy, yielding an estimated initial fractional coverage of 0.25 and a final coverage of 0.5. Hybridization on the nanotube surface is confirmed using Forster resonance energy transfer of fluorophore-labeled DNA oligonucleotides. This detection is enabled through a new technique to suspend SWNTs using adsorption of single-stranded DNA and subsequent removal of free DNA from solution. While the kinetics of free DNA hybridization are relatively fast (<10 min), the kinetics of the process on SWNTs are slower under comparable conditions, reaching steady state after 13 ... [truncated at 150 words]
Vest RS, Wallis R, Jensen LB, Hawes AC, Callister J, Brimhall B, Judd AM, Bell JD.
Use of steady-state Laurdan fluorescence to detect changes in liquid ordered phases in human erythrocyte membranes.
J Membrane Biol. 2006; 211(1): 15-25.In artificial phospholipid bilayers, dual measurements of laurdan steady-state anisotropy and emission spectra can be used to identify the presence of liquid ordered phases. Human erythrocytes were used as a model to test whether similar measurements could be applied to biological samples. Specifically, laurdan anisotropy and emission spectra were obtained from native erythrocytes before and after treatment with calcium ionophore and from the microvesicles (known to be enriched in liquid ordered domains) shed from the cells during calcium entry. Spectral and anisotropy data were consistent with an increased order and reduced fluidity of erythrocyte membrane lipids upon ionophore treatment. Microvesicle membranes appeared more ordered than native erythrocytes and similar to ionophore-treated cells based on laurdan emission. In contrast, the anisotropy value was lower in microvesicles compared to ionophore-treated cells, suggesting greater probe mobility. Parallel measurements of diphenylhexatriene anisotropy corroborated the laurdan data. These results were consistent with the liquid ordered ... [truncated at 150 words]
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Fluorescent DNAzyme biosensors for metal ions based on catalytic molecular beacons.
Fluorescent Energy Transfer Nucleic Acid Probes Designs and Protocols (Methods in Molecular Biology, Vol. 335). By VV Didenko (Editors). Humana Press, pp. 275-288, 2006. ISBN: 9781588293800In this chapter, methods for designing metal ion sensors using fluorophore- and quencher-labeled DNAzymes are discussed. In contrast to the classical molecular beacon method based on binding, the methods described here utilize catalytic cleavage to release the fluorophore for detection and quantification, making it possible to take advantage of catalytic turnovers for signal amplification. Unlike classical molecular beacons that detect only nucleic acids, catalytic molecular beacons can be applied to different DNAzymes to detect a broad range of analytes. The methods described are based on the finding that almost all known trans-cleaving DNAzymes share a similar structure comprised of a catalytic DNAzyme core flanked by two substrate recognition arms. Using a typical DNAzyme called the "8-17" DNAzyme as an example, the design of highly sensitive and selective Pb2+ sensors is described in detail. The initial design employs a single fluorophore-quencher pair in close proximity, with the fluorophore on the 5'-end ... [truncated at 150 words]
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Multi-fluorophore fluorescence resonance energy transfer for probing nucleic acids structure and folding.
Fluorescent Energy Transfer Nucleic Acid Probes Designs and Protocols (Methods in Molecular Biology, Vol. 335). By VV Didenko (Editors). Humana Press, pp. 257-71, 2006. ISBN: 9781588293800Fluorescence resonance energy transfer (FRET) is a widely used technique to study the structure and dynamics of nucleic acids in solution. Such a technique often uses only one donor fluorophore and one acceptor fluorophore to probe the distance and its changes between the two labeled sites. To fully understand molecules with complicated structures, such as three- or four-way DNA junctions, several dual-fluorophore experiments have to be performed. Here, we describe an emerging alternative technique using multi-fluorophore FRET, in which simultaneous labeling of one molecule with several different fluorophores is performed to acquire all the distance information in a single experiment. This method decreases the number of experiments necessary to perform and increases the consistency of the results. In this chapter, FRET study of a tri-fluorophore-labeled DNAzyme serves as an example to illustrate the design of multi-fluorophore FRET experiments and the related data processing and analysis. The (ratio)A method used to ... [truncated at 150 words]
Madhavan N, Robert EC, Gin MS.
A highly active anion-selective aminocyclodextrin ion channel.
Angew Chem Int Ed Engl. 2005; 44(46): 7584-7.Ion-channel proteins are molecular devices found in nature that mediate the transport of charged species across cell membranes. Various functions in the human body, such as nerve and muscle excitation, hormonal secretion, cell proliferation, and homeostasis, are regulated by ion channels. The efficient functioning of ion-transport machinery in nature has led to considerable interest in understanding the mechanisms by which these proteins mediate selective, regulated transmembrane ion transport. Furthermore, the ability of ion channels to effect electrical signaling under aqueous saline conditions has inspired their use as biosensors, therapeutic agents,[4] and other useful materials. Despite the potential utility of controlled ion transport, the relative instability of proteins in vitro will likely prohibit their use in commercial applications. Advances in the synthesis of more robust artificial channels provide a viable solution to the problem of ion-channel stability, thus enabling the construction of biomimetic signaling components.
Woodyer R, Zhao H, van der Donk WA.
Mechanistic investigation of a highly active phosphite dehydrogenase mutant and its application for NADPH regeneration.
FEBS J. 2005; 272(15): 3816-27.NAD(P)H regeneration is important for biocatalytic reactions that require these costly cofactors. A mutant phosphite dehydrogenase (PTDH-E175A/A176R) that utilizes both NAD and NADP efficiently is a very promising system for NAD(P)H regeneration. In this work, both the kinetic mechanism and practical application of PTDH-E175A/A176R were investigated for better understanding of the enzyme and to provide a basis for future optimization. Kinetic isotope effect studies with PTDH-E175A/A176R showed that the hydride transfer step is (partially) rate determining with both NAD and NADP giving (D)V values of 2.2 and 1.7, respectively, and (D)V/K(m,phosphite) values of 1.9 and 1.7, respectively. To better comprehend the relaxed cofactor specificity, the cofactor dissociation constants were determined utilizing tryptophan intrinsic fluorescence quenching. The dissociation constants of NAD and NADP with PTDH-E175A/A176R were 53 and 1.9 microm, respectively, while those of the products NADH and NADPH were 17.4 and 1.22 microm, respectively. Using sulfite as a substrate mimic, ... [truncated at 150 words]
Aathimanikandan SV, Sandanaraj BS, Arges CG, Bardeen CJ, Thayumanavan S.
Effect of guest molecule flexibility in access to dendritic interiors.
Org Lett. 2005; 7(14): 2809-12.[structure: see text] Dendrimers are attractive scaffolds for catalysis, since catalytic sites can be isolated and the catalysts are recoverable and reusable. Herein, we show that conformationally constrained molecules have better access to dendritic cores compared to the more flexible counterparts. The results reported here should have implications in utilizing dendrimers as scaffolds for artificial selectivity in catalysis.
Breusegem SY, Halaihel N, Inoue M, Zajicek HK, Lederer E, Barry NP, Sorribas V, Levi M.
Acute and chronic changes in cholesterol modulate Na-Pi cotransport activity in OK cells.
Am J Physiol Renal Physiol. 2005; 289(1): F154-65.We previously showed an inverse correlation between membrane cholesterol content and Na-P(i) cotransport activity during the aging process and adaptation to alterations in dietary P(i) in the rat (Levi M, Jameson DM, and van der Meer BW. Am J Physiol Renal Fluid Electrolyte Physiol 256: F85-F94, 1989). The purpose of the present study was to determine whether alterations in cholesterol content per se modulate Na-P(i) cotransport activity and apical membrane Na-P(i) protein expression in opossum kidney (OK) cells. Acute cholesterol depletion achieved with beta-methyl cyclodextrin (beta-MCD) resulted in a significant increase in Na-P(i) cotransport activity accompanied by a moderate increase in apical membrane Na-P(i) protein abundance and no alteration of total cellular Na-P(i) protein abundance. Conversely, acute cholesterol enrichment achieved with beta-MCD/cholesterol resulted in a significant decrease in Na-P(i) cotransport activity with a moderate decrease in apical membrane Na-Pi protein abundance and no change of the total cellular Na-P(i) protein ... [truncated at 150 words]
Relyea HA, Vrtis JM, Woodyer R, Rimkus SA, van der Donk WA.
Inhibition and pH dependence of phosphite dehydrogenase.
Biochemistry. 2005; 44(17): 6640-9.Phosphite dehydrogenase (PTDH) catalyzes the NAD-dependent oxidation of phosphite to phosphate, a reaction that is 15 kcal/mol exergonic. The enzyme belongs to the family of D-hydroxy acid dehydrogenases. Five other family members that were analyzed do not catalyze the oxidation of phosphite, ruling out the possibility that this is a ubiquitous activity of these proteins. PTDH does not accept any alternative substrates such as thiophosphite, hydrated aldehydes, and methylphosphinate, and potential small nucleophiles such as hydroxylamine, fluoride, methanol, and trifluoromethanol do not compete with water in the displacement of the hydride from phosphite. The pH dependence of k(cat)/K(m,phosphite) is bell-shaped with a pK(a) of 6.8 for the acidic limb and a pK(a) of 7.8 for the basic limb. The pK(a) of 6.8 is assigned to the second deprotonation of phosphite. However, whether the dianionic form of phosphite is the true substrate is not clear since a reverse protonation mechanism is ... [truncated at 150 words]
Jensen LB, Burgess NK, Gonda DD, Spencer E, Wilson-Ashworth HA, Driscoll E, Vu MP, Fairbourn JL, Judd AM, Bell JD.
Mechanisms governing the level of susceptibility of erythrocyte membranes to secretory phospholipase A2.
Biophys J. 2005; 88(4): 2692-2705. PMCID: PMC1305365Although cell membranes normally resist the hydrolytic action of secretory phospholipase A(2) (sPLA(2)), they become susceptible during apoptosis or after cellular trauma. Experimentally, susceptibility to the enzyme can be induced by loading cells with calcium. In human erythrocytes, the ability of the calcium ionophore to cause susceptibility depends on temperature, occurring best above approximately 35 degrees C. Considerable evidence from experiments with artificial bilayers suggests that hydrolysis of membrane lipids requires two steps. First, the enzyme adsorbs to the membrane surface, and second, a phospholipid diffuses from the membrane into the active site of the adsorbed enzyme. Analysis of kinetic experiments suggested that this mechanism can explain the action of sPLA(2) on erythrocyte membranes and that temperature and calcium loading promote the second step. This conclusion was further supported by binding experiments and assessment of membrane lipid packing. The adsorption of fluorescent-labeled sPLA(2) was insensitive to either temperature or ionophore ... [truncated at 150 words]
Woodyer R, Wheatley JL, Relyea HA, Rimkus SA, van der Donk WA.
Site-directed mutagenesis of active site residues of phosphite dehydrogenase.
Biochemistry. 2005; 44(12): 4765-74.Phosphite dehydrogenase (PTDH) catalyzes the unusual oxidation of phosphite to phosphate with the concomitant reduction of NAD(+) to NADH. PTDH shares significant amino acid sequence similarity with D-hydroxy acid dehydrogenases (DHs), including strongly conserved catalytic residues His292, Glu266, and Arg237. Site-directed mutagenesis studies corroborate the essential role of His292 as all mutants of this residue were completely inactive. Histidine-selective inactivation studies with diethyl pyrocarbonate provide further evidence regarding the importance of His292. This residue is most likely the active site base that deprotonates the water nucleophile. Kinetic analysis of mutants in which Arg237 was changed to Leu, Lys, His, and Gln revealed that Arg237 is involved in substrate binding. These results agree with the typical role of this residue in D-hydroxy acid DHs. However, Glu266 does not play the typical role of increasing the pK(a) of His292 to enhance substrate binding and catalysis as the Glu266Gln mutant displayed an increased ... [truncated at 150 words]
Arnulphi C, Jin L, Tricerri MA, Jonas A.
Enthalpy-driven apolipoprotein A-I and lipid bilayer interaction indicating protein penetration upon lipid binding.
Biochemistry. 2004; 43(38): 12258-64.The interaction of lipid-free apolipoprotein A-I (apoA-I) with small unilamellar vesicles (SUVs) of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with and without free cholesterol (FC) was studied by isothermal titration calorimetry and circular dichroism spectroscopy. Parameters reported are the affinity constant (K(a)), the number of protein molecules bound per vesicle (n), enthalpy change (DeltaH degrees), entropy change (DeltaS degrees ), and the heat capacity change (DeltaC(p) degrees). The binding process of apoA-I to SUVs of POPC plus 0-20% (mole) FC was exothermic between 15 and 37 degrees C studied, accompanied by a small negative entropy change, making enthalpy the main driving force of the interaction. The presence of cholesterol in the vesicles increased the binding affinity and the alpha-helix content of apoA-I but lowered the number of apoA-I bound per vesicle and the enthalpy and entropy changes per bound apoA-I. Binding affinity and stoichiometry were essentially invariant of temperature for binding to SUVs of ... [truncated at 150 words]
Payton JE, Perrin RJ, Woods WS, George JM.
Structural determinants of PLD2 inhibition by α-synuclein.
J Mol Biol. 2004; 337(4): 1001-9.The presynaptic protein α-synuclein has been implicated in both neuronal plasticity and neurodegenerative disease, but its normal function remains unclear. We described the induction of an amphipathic α-helix at the N terminus (exons 2-4) of α-synuclein upon exposure to phospholipid vesicles, and hypothesized that lipid-binding might serve as a functional switch by stabilizing α-synuclein in an active (α-helical) conformation. Others have shown that alpha and β-synucleins inhibit phospholipase D (PLD), an enzyme involved in lipid-mediated signaling cascades and vesicle trafficking. Here, we report that all three naturally occurring synuclein isoforms (α, β, and γ-synuclein) are similarly effective inhibitors of PLD2 in vitro, as is the Parkinson's disease-associated mutant A30P. The PD-associated mutant A53T, however, is a more potent inhibitor of PLD2 than is wild-type α-synuclein. We analyze mutations of the alpha-synuclein protein to identify critical determinants of human PLD2 inhibition in vitro. Deletion of residues 56-102 (exon 4) decreases PLD2 ... [truncated at 150 words]
Vest RS, Gonzales LJ, Permann SA, Spencer E, Hansen LD, Judd AM, Bell JD.
Divalent cations increase lipid order in erythrocytes and susceptibility to secretory phospholipase A2.
Biophys J. 2004; 86(4): 2251-2260. PMCID: PMC1304075Elevated concentrations of intracellular calcium in erythrocytes increase membrane order and susceptibility to secretory phospholipase A2. We hypothesize that calcium aids the formation of domains of ordered lipids within erythrocyte membranes by interacting directly with the inner leaflet of the cell membrane. The interface of these domains with regions of more fluid lipids may create an environment with weakened neighbor-neighbor interactions that would facilitate phospholipid migration into the active site of bound secretory phospholipase A2. This hypothesis was investigated by determining the effects of seven other divalent ions on erythrocyte membrane properties. Changes in membrane order were assessed with steady-state fluorescence spectroscopy and two-photon microscopy with an environment-sensitive probe, laurdan. Each ion increased apparent membrane order in model membranes and in erythrocytes when introduced with an ionophore, suggesting that direct binding to the inner face of the membrane accounts for the effects of calcium on membrane fluidity. Furthermore, the degree ... [truncated at 150 words]
Murphy MC, Rasnik I, Cheng W, Lohman TM, Ha T.
Probing single-stranded DNA conformational flexibility using fluorescence spectroscopy.
Biophys J. 2004; 86(4): 2530-2537. PMCID: PMC1304100Single-stranded DNA (ssDNA) is an essential intermediate in various DNA metabolic processes and interacts with a large number of proteins. Due to its flexibility, the conformations of ssDNA in solution can only be described using statistical approaches, such as flexibly jointed or worm-like chain models. However, there is limited data available to assess such models quantitatively, especially for describing the flexibility of short ssDNA and RNA. To address this issue, we performed FRET studies of a series of oligodeoxythymidylates, (dT)N, over a wide range of salt concentrations and chain lengths (10 ≤ N ≤ 70 nucleotides), which provide systematic constraints for testing theoretical models. Unlike in mechanical studies where available ssDNA conformations are averaged out during the time it takes to perform measurements, fluorescence lifetimes may act here as an internal clock that influences fluorescence signals depending on how fast the ssDNA conformations fluctuate. A reasonably good agreement could be ... [truncated at 150 words]
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Improving fluorescent DNAzyme biosensors by combining inter- and intramolecular quenchers.
Anal Chem. 2003; 75(23): 6666-72.A previously reported DNAzyme-based biosensor for Pb(2+) has shown high sensitivity and selectivity at 4 degrees C. In the system, the substrate and the enzyme strand of the DNAzyme are labeled with a fluorophore and a quencher, respectively. In the presence of Pb(2+), the substrate strand is cleaved by the enzyme strand, and the release of the cleaved fragment results in significant fluorescence increase. However, the performance of the sensor decreases considerably if the temperature is raised to room temperature because of high background fluorescence. A careful analysis of the sensor system, including measurement of the melting curve and fluorescence resonance energy-transfer (FRET) study of the free substrate, suggests that a fraction of the fluorophore-labeled substrate strand is dissociated from the enzyme strand, resulting in elevated background fluorescence signals at room temperature. To overcome this problem, we designed a new sensor system by introducing both inter- and intramolecular quenchers. The ... [truncated at 150 words]
Düzgünes N, Bagatolli LA, Meers P, Oh YK, Straubinger RM.
Fluorescence methods in liposome research.
Liposomes 2nd ed: A Practical Approach (The Practical Approach Series, Vol. 264). By V Torchilin and V Weissig (Editors). Oxford University Press, USA, pp. 105-147, 2003. ISBN: 9780199636549Lu Y, Liu J, Li J, Bruesehoff PJ, Pavot CMB, Brown AK.
New highly sensitive and selective catalytic DNA biosensors for metal ions.
Biosens Bioelectr. 2003; 18(5-6): 529-40.While remarkable progress has been made in developing sensors for metal ions such as Ca(II) and Zn(II), designing and synthesizing sensitive and selective metal ion sensors remains a significant challenge. Perhaps the biggest challenge is the design and synthesis of a sensor capable of specific and strong metal binding. Since our knowledge about the construction of metal-binding sites in general is limited, searching for sensors in a combinatorial way is of significant value. Therefore, we have been able to use a combinatorial method called in vitro selection to obtain catalytic DNA that can bind a metal ion of choice strongly and specifically. The metal ion selectivity of the catalytic DNA was further improved using a 'negative selection' strategy where catalytic DNA that are selective for competing metal ions are discarded in the in vitro selection processes. By labeling the resulting catalytic DNA with a fluorophore/quencher pair, we have made a ... [truncated at 150 words]
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Direct observation of lipid domains in free standing bilayers: from simple to complex lipid mixtures.
Chem Phys Lipids. 2003; 122(1-2): 137-45.The direct observation of temperature-dependent lipid phase equilibria, using two-photon excitation fluorescence microscopy on giant unilamellar vesicles (GUVs) composed of different lipid mixtures, provides novel information about the physical characteristics of lipid domain coexistence. Physical characteristics such as shape, size, and time evolution of different lipid domains are not directly accessible from the traditional experimental approaches that employ either small and large unilamellar vesicles or multilamellar vesicles. In this short presentation, I will address the most relevant findings reported from our laboratory, regarding the direct observation of lipid domain coexistence at the level of single vesicles in artificial and natural lipid mixtures. In addition, key points concerning our experimental approach will be discussed. The unique advantages of the fluorescent probe 6-dodecanoyl-2-dimethylamino-naphthalene (LAURDAN) under the two-photon excitation fluorescence microscopy will be particularly addressed, especially, the possibility to obtain information about the phase-state of different lipid domains directly from the fluorescent images.
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FRET study of a trifluorophore-labeled DNAzyme.
J Am Chem Soc. 2002; 124(51): 15208-16.A fluorescence resonance energy transfer (FRET) study of biomolecules typically employs two fluorophores. The increasing number of branches and complexity of biomolecules call for simultaneously monitoring structures and dynamics of several branches in a single system. Furthermore, despite recent studies that show DNAzymes can be a stable and cost-effective alternative to protein and ribozymes for pharmaceutical and biotechnological applications, no FRET study of DNAzymes has been reported. Here, we describe the FRET study of a trifluorophore-labeled "8-17" DNAzyme, in which each of the three branches is labeled with a different fluorophore. From the study, we found that the (ratio)(A) method that has been commonly used in dual-fluorophore-labeled systems is also applicable to trifluorophore-labeled systems. However, while both FRET efficiency and fluorophore-to-fluorophore distance can be used to measure FRET in dual-fluorophore-labeled systems, only the average distance should be used in trifluorophore-labeled systems. The ability to monitor all three branches in a ... [truncated at 150 words]
da Poian AT, Johnson JE, Silva JL.
Protein-RNA interactions and virus stability as probed by the dynamics of Tryptophan side chains.
J Biol Chem. 2002; 277(49): 47596-47602.The correlation between dynamics and stability of icosahedral viruses was studied by steady-state and time-resolved fluorescence approaches. We compared the environment and dynamics of tryptophan side chains of empty capsids and ribonucleoprotein particles of two icosahedral viruses from the comovirus group: cowpea mosaic virus (CPMV) and bean pod mottle virus (BPMV). We found a great difference between tryptophan fluorescence emission spectra of the ribonucleoprotein particles and the empty capsids of BPMV. For CPMV, time-resolved fluorescence revealed differences in the tryptophan environments of the capsid protein. The excited-state lifetimes of tryptophan residues were significantly modified by the presence of RNA in the capsid. More than half of the emission of the tryptophans in the ribonucleoprotein particles of CPMV originates from a single exponential decay that can be explained by a similar, nonpolar environment in the local structure of most of the tryptophans, even though they are physically located in different regions ... [truncated at 150 words]
Snow CD, Nguyen H, Pande VS, Gruebele M.
Absolute comparison of simulated and experimental protein-folding dynamics.
Nature. 2002; 420(6911): 102-106.Protein folding is difficult to simulate with classical molecular dynamics. Secondary structure motifs such as alpha-helices and beta-hairpins can form in 0.1-10 micros (ref. 1), whereas small proteins have been shown to fold completely in tens of microseconds. The longest folding simulation to date is a single 1- micro s simulation of the villin headpiece; however, such single runs may miss many features of the folding process as it is a heterogeneous reaction involving an ensemble of transition states. Here, we have used a distributed computing implementation to produce tens of thousands of 5-20-ns trajectories (700 micros) to simulate mutants of the designed mini-protein BBA5. The fast relaxation dynamics these predict were compared with the results of laser temperature-jump experiments. Our computational predictions are in excellent agreement with the experimentally determined mean folding times and equilibrium constants. The rapid folding of BBA5 is due to the swift formation of secondary ... [truncated at 150 words]
Smith A, Chaïeb S, Aql Aa, Alsalhi M, Nayfeh MH.
Observation of assembly of fluorescent Si nanoparticles under the influence of electric current.
J Nanosci Nanotechnol. 2002; 2(5): 471-3.A silicon substrate patterned by an oxide is immersed in an alcohol solution of low-doped 1-nm Si nanoparticles. Reverse biasing draws particles to the substrate, mostly along the conducting current paths. Scanning electron and fluorescence microscopy show a tree-like network on the substrate. Avoidance of closed loops and preference for an angle of branching of 90 degrees-120 degrees are observed. The building block of the tree network is not individual particles but spherical particle aggregates approximately 150 nm in diameter.
Harris FM, Best KB, Bell JD.
Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order.
Biochim Biophys Acta. 2002; 1565(1): 123-128.Laurdan is a fluorescent probe that detects changes in membrane phase properties through its sensitivity to the polarity of its environment in the bilayer. Variations in membrane water content cause shifts in the laurdan emission spectrum, which are quantified by calculating the generalized polarization (GP). We tested whether laurdan fluorescence could be used to distinguish differences in phospholipid order from changes in membrane fluidity by examining the temperature dependence of laurdan GP and fluorescence anisotropy in dipalmitoylphosphatidylcholine (DPPC) vesicles. The phase transition from the solid ordered phase to the liquid disordered phase was observed as a decrease in laurdan GP values from 0.7 to −0.14 and a reduction in anisotropy from 0.25 to 0.12. Inclusion of various amounts of cholesterol in the membranes to generate a liquid ordered phase caused an increase in the apparent melting temperature detected by laurdan GP. In contrast, cholesterol decreased the apparent melting temperature estimated ... [truncated at 150 words]
Bagatolli LA, Binns DD, Jameson DM, Albanesi JP.
Activation of dynamin II by POPC in giant unilamellar vesicles: a two-photon fluorescence microscopy study.
J Protein Chem. 2002; 21(6): 383-391.The interaction of dynamin II with giant unilamellar vesicles was studied using two-photon fluorescence microscopy. Dynamin II, labeled with fluorescein, was injected into a microscope chamber containing giant unilamellar vesicles, which were composed of either pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of POPC and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Binding of the fluorescent dynamin II to giant unilamellar vesicles, in the presence and absence of PI(4,5)P2, was directly observed using two-photon fluorescence microscopy. This binding was also visualized using the fluorescent N-methylanthraniloyl guanosine 5'-[gamma-thio]triphosphate analogue. The membrane probe 6-dodecanoyl-2-dimethylamine-naphthalene was used to monitor the physical state of the lipid in the giant unilamellar vesicles in the absence and presence of dynamin. A surprising finding was the fact that dynamin II bound to vesicles in the absence of PI(4,5)P2. Activation of the GTPase activity of dynamin II by pure POPC was then shown.
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The energy efficiency of formation of photons, radicals and ions during single-bubble cavitation.
Nature. 2002; 418(6896): 394-397.It is extremely difficult to perform a quantitative analysis of the chemistry associated with multibubble cavitation: unknown parameters include the number of active bubbles, the acoustic pressure acting on each bubble and the bubble size distribution. Single-bubble sonoluminescence (characterized by the emission of picosecond flashes of light) results from nonlinear pulsations of an isolated vapour-gas bubble in an acoustic field. Although the latter offers a much simpler environment in which to study the chemical activity of cavitation, quantitative measurements have been hindered by the tiny amount of reacting gas within a single bubble (typically <10(-13) mol). Here we demonstrate the existence of chemical reactions within a single cavitating bubble, and quantify the sources of energy dissipation during bubble collapse. We measure the yields of nitrite ions, hydroxyl radicals and photons. The energy efficiency of hydroxyl radical formation is comparable to that in multibubble cavitation, but the energy efficiency of light ... [truncated at 150 words]
Nag K, Pao JS, Harbottle RR, Possmayer F, Petersen NO, Bagatolli LA.
Segregation of saturated chain lipids in pulmonary surfactant films and bilayers.
Biophys J. 2002; 82(4): 2041-51. PMCID: PMC1301999The physical properties of organized system (bilayers and monolayers at the air water interface) composed of bovine lipid extract surfactant (BLES) were studied using correlated experimental techniques. 6-Dodecanoyl-2-dimethylamino-naphthalene (LAURDAN)-labeled giant unilamelar vesicles (mean diameter approximately 30 microm) composed of BLES were observed at different temperatures using two-photon fluorescence microscopy. As the temperature was decreased, dark domains (gel-like) appeared at physiological temperature (37 degrees C) on the surface of BLES giant unilamelar vesicles. The LAURDAN two-photon fluorescent images show that the gel-like domains span the lipid bilayer. Quantitative analysis of the LAURDAN generalized polarization function suggests the presence of a gel/fluid phase coexistence between 37 degrees C to 20 degrees C with low compositional and energetic differences between the coexisting phases. Interestingly, the microscopic scenario of the phase coexistence observed below 20 degrees C shows different domain's shape compared with that observed between 37 degrees C to 20 degrees C, suggesting ... [truncated at 150 words]
Agree AKB, Tricerri MA, Arnvig-McGuire K, Tian Sm, Jonas A.
Folding and stability of the C-terminal half of apolipoprotein A-I examined with a Cys-specific fluorescence probe.
Biochim Biophys Acta. 2002; 1594(2): 286-96.Apolipoprotein A-I (apoA-I) has important physiologic roles in reverse cholesterol transport, as a component of HDL; however, apoA-I also exists in lipid-poor or lipid-free forms that are key intermediates in HDL metabolism and acceptors of lipids from cells. The aim of this study was to examine the structure and stability of the central and C-terminal regions of lipid-free apoA-I. To this end, five Cys mutants of proapoA-I were constructed and expressed in Escherichia coli: V119C, A124C, A154C, A190C, and A232C. These mutants were specifically labeled with 6-acryloyl-2-dimethylaminonaphthalene (acrylodan, AC) and were examined by CD spectroscopy and a variety of fluorescence methods. The results showed that the introduction of Cys residues and their covalent labeling with AC did not affect the overall structure and stability of apoA-I. However, AC fluorescence properties revealed that different segments of the central and C-terminal half of apoA-I have distinct folding and stability properties. From fluorescence ... [truncated at 150 words]
Sotomayor CP, Aguilar LF, Cuevas FJ, Helms MK, Jameson DM.
Modulation of pig kidney Na+/K+-ATPase activity by cholesterol: role of hydration.
Biochemistry. 2000; 39(35): 10928-35.Cholesterol is known to affect the activity of membrane-bound enzymes, including Na(+)/K(+)-ATPase. To gain insight into the mechanism of cholesterol's effect, we have used various hydrophobic fluorescent probes which insert into different regions of the membrane bilayer and report on the degree of hydration of their environment. Specifially, we have measured the generalized polarization of Laurdan and the lifetime of DPH and derivatives of DPH inserted into membranes from pig kidneys enriched in Na(+)/K(+)-ATPase. Spectral measurements were also carried out on these membranes after modification of their cholesterol content. The generalized polarization of Laurdan increased with increasing cholesterol, showing an abrupt modification at the native cholesterol content. The fluorescence lifetimes of DPH and the DPH derivatives were analyzed using a distribution model. The center value of these lifetime distributions and their widths also changed with increasing cholesterol. One DPH derivative, DPH-PC, showed a minimum value for the lifetime center at ... [truncated at 150 words]
Bondosa SE, Sligar SG, Jonas J.
High-pressure denaturation of apomyoglobin.
Biochim Biophys Acta. 2000; 1480(1-2): 353-64.The pressure denaturation of wild type and mutant apomyoglobin (apoMb) was investigated using a high-pressure, high-resolution nuclear magnetic resonance and high-pressure fluorescence techniques. Wild type apoMb is resistant to pressures up to 80 MPa, and denatures to a high-pressure intermediate, I(p), between 80 and 200 MPa. A further increase of pressure to 500 MPa results in denaturation of the intermediate. The two tryptophans, both in the A helix, remain sequestered from solvent in the high-pressure intermediate, which retains some native NOESY cross peaks in the AGH core as well as between F33 and F43. High-pressure fluorescence shows that the tryptophans remain inaccessible to solvent in the I(p) state. Thus the high-pressure intermediate has some structural properties in common with the apoMb I(2) acid intermediate. The resistance of the AGH core to pressures up to 200 MPa provides further evidence that the intrinsic stability of these alpha-helices is responsible for their ... [truncated at 150 words]
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Application of near infra-red spectroscopy to the non-invasive measurement of relevant physiological parameters: the blood flow and the oxygen consumption in the skeletal muscle.
PhD in Physics, University of Bari, Italy, 2000.
Advisor: Pietro Mario Lugarà
Xiao M, Li H, Snyder GE, Cooke R, Yount RG, Selvin PR.
Conformational changes between the active-site and regulatory light chain of myosin as determined by luminescence resonance energy transfer: the effect of nucleotides and actin.
Proc Natl Acad Sci USA. 1998; 95(26): 15309-15314. PMCID: PMC28039Myosin is thought to generate movement of actin filaments via a conformational change between its light-chain domain and its catalytic domain that is driven by the binding of nucleotides and actin. To monitor this change, we have measured distances between a gizzard regulatory light chain (Cys 108) and the active site (near or at Trp 130) of skeletal myosin subfragment 1 (S1) by using luminescence resonance energy transfer and a photoaffinity ATP-lanthanide analog. The technique allows relatively long distances to be measured, and the label enables site-specific attachment at the active-site with only modest affect on myosin’s enzymology. The distance between these sites is 66.8 ± 2.3 Å when the nucleotide is ADP and is unchanged on binding to actin. The distance decreases slightly with ADP-BeF3, (−1.6 ± 0.3 Å) and more significantly with ADP-AlF4 (−4.6 ± 0.2 Å). During steady-state hydrolysis of ATP, the distance is temperature-dependent, becoming shorter ... [truncated at 150 words]
Blackman SM, Piston DW, Beth AH.
Oligomeric state of human erythrocyte band 3 measured by fluorescence resonance energy homotransfer.
Biophys J. 1998; 75(2): 1117-1130. PMCID: PMC1299786The oligomeric state of the erythrocyte anion exchange protein, band 3, has been assayed by resonance energy homotransfer. Homotransfer between oligomeric subunits, labeled with eosin-5-maleimide at Lys430 in the transmembrane domain, has been demonstrated by steady-state and time-resolved fluorescence spectroscopy, and is readily observed by its depolarization of the eosin fluorescence. Polarized fluorescence measurements of HPLC-purified band 3 oligomers indicate that eosin homotransfer increases progressively with increasing species size. This shows that homotransfer also occurs between labeled band 3 dimers as well as within the dimers, making fluorescence anisotropy measurements sensitive to band 3 self-association. Treatment of ghost membranes with either Zn2+ or melittin, agents that cluster band 3, significantly decreases the anisotropy as a result of the increased homotransfer within the band 3 clusters. By comparison with the anisotropy of species of known oligomeric state, the anisotropy of erythrocyte ghost membranes at 37 degrees C is consistent with dimeric ... [truncated at 150 words]
Helms MK, Malencik DA, Anderson SR.
Flexibility involving the intermolecular dityrosyl cross-links of enzymatically polymerized calmodulin.
Biochemistry. 1998; 37(23): 8378-84.The role of dityrosine as a fluorescent crossbridge between adjacent calmodulin molecules within the high molecular mass polymers that are generated by Arthromyces peroxidase-catalyzed cross-linking [Malencik, D. A., and Anderson, S. R. (1996) Biochemistry 35, 4375] has been examined in frequency domain fluorescence anisotropy studies. Measurements on a polymer fraction possessing a range of molecular masses > 96 000 in NaDodSO4 polyacrylamide gel electrophoresis demonstrate predominating fast local rotations involving the dityrosyl moieties. Normal distribution analyses of the results show peak rotational correlation times of 0.6 ns (zero Ca2+) and 1.2 ns (+Ca2+), values that are smaller than the principal correlation times determined for the global rotation of the free calmodulin monomer in either the presence or absence of Ca2+. The intermolecularly cross-linked segments of the polymers retain a degree of the mobility that is characteristic of the tyrosine-containing sequences of native calmodulin. The half-widths of the normal distribution curves ... [truncated at 150 words]
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Escherichia coli cAMP receptor protein-DNA complexes. 2. Structural asymmetry of DNA bending.
Biochemistry. 1998; 37(15): 5201-10.The effect of DNA sequence variability and the degree of cyclic AMP receptor protein (CRP)-induced bending of the flanking ends of fluorescently labeled DNA were investigated by steady-state fluorescence and differential phase polarization studies in the presence and absence of CRP. Six sequences, including the primary CRP binding sites of lac P1 (class I) and gal P1 (class II), were studied. Excitation and emission spectra of CPM-DNA upon binding CRP were observed to be qualitatively similar to one another, regardless of the CRP binding site sequence examined or the location of the probe. This result implies that the probe is not interacting with the protein. However, the magnitude of the changes in the fluorescence intensities of sensitized emission spectra of CPM-DNA is apparently dependent on the DNA sequence, indicating that the environments of the flanking ends of DNA may be different from one another in the protein-DNA complex. Differential phase ... [truncated at 150 words]
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A flow cytometric method for measurement of intracellular chloride concentration in lymphocytes using the halide-specific probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ).
Cytometry. 1997; 28(4): 316-22.A flow cytometry method using the halide-specific fluorescent dye, 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), has been developed to measure intracellular chloride concentration in single cells. Collisions with chloride quench the fluorescence of SPQ, making it possible to relate the measured fluorescence intensity to chloride concentration with a Stern-Volmer equation. To demonstrate the method, porcine lymphocytes were loaded in vitro, using a hypotonic method, with 5 mM SPQ. Fluorescence excitation was provided by a UV laser and the fluorescence emission intensity at 485 nm was recorded. Calibration was performed by using 7 microM nigericin (a K/H antiporter) and 10 microM tributyltin (a Cl/OH antiporter) to equilibrate the concentrations of intracellular and extracellular chloride. Calibration measurements were made for chloride concentrations between 0 mM and 140 mM. The calibration produced a Stern-Volmer quenching constant of 16.2 M(-1) which was used to relate measured cell fluorescence to intracellular chloride concentration. The intracellular chloride concentration for ... [truncated at 150 words]
Helms MK, Petersen CE, Bhagavan NV, Jameson DM.
Time-resolved fluorescence studies on site-directed mutants of human serum albumin.
FEBS Lett. 1997; 408(1): 67-70.Human serum albumin (HSA) contains a single tryptophan residue at position 214. The emission properties of tryptophan 214 from recombinant albumins, namely, normal HSA, FDH-HSA and a methionine 218 HSA were examined. In all cases, the excited state lifetimes were best described by a two component model consisting mainly of a Lorentzian distribution. The centers of these distributions were 5.60 ns for HSA, 4.23 ns for FDH-HSA, and 6.08 ns for Met-218 HSA. The global rotational correlation times of the three HSAs were near 41 ns while the amplitude and rate of the local motion varied. These changes in the lifetimes and mobilities suggest perturbation in the local protein environment near tryptophan 214 as a consequence of the amino acid substitutions.
Weaver DJ Jr, Durack G, Voss EW Jr.
Analysis of the intracellular processing of proteins: application of fluorescence polarization and a novel fluorescent probe.
Cytometry. 1997; 28(1): 25-35.Previous studies indicated that fluorescein derivatized bovine serum albumin was an ideal probe to monitor the time-dependent kinetics of antigen processing in the murine macrophage cell line J774. Whereas previous work focused on fluorescence intensity measurements, the present study relied on fluorescence polarization to dissect the local environment of the fluorescent hapten-protein within the endocytic system of the cell. A steady increase in both fluorescence intensity and fluorescence polarization of the cell population was detected for the first 100 min. However, at 100 min, a plateau in both fluorescence intensity and polarization was observed and was followed by a decrease in fluorescence polarization and a corresponding increase in fluorescence intensity. Western blot analyses revealed that the decrease in fluorescence polarization was due to proteolytic degradation of the probe within the cell. Using a combination of in vitro experiments and an additional fluorescent probe, it was determined that the initial increase ... [truncated at 150 words]
Carrero J, Mallender WD, Voss EW Jr.
Anti-metatype antibody stabilization of Fv 4-4-20 variable domain dynamics.
J Biol Chem. 1996; 271(19): 11247-52.Anti-metatype (anti-Met) antibodies are immunoglobulins that specifically recognize and stabilize antibodies in their liganded or metatypic state, but lack specificity for either the hapten or the unliganded antibody. Autologous anti-Met antibodies were previously observed in vivo, suggesting that a metatypic autoantibody response could play a physiological role in the immune network, e.g. controlling the clearance of immune complexes from circulation. The first elicited anti-Met antibodies were against the fluorescein-liganded high affinity murine anti-fluorescein monoclonal antibody 4-4-20. The fluorescein-hapten system has proved to be an invaluable tool for both the recognition and characterization of the metatypic response by utilization of its spectral properties. In this investigation, hydrostatic pressure measurements, in conjunction with fluorescence spectroscopy, were performed on the recombinant Fv derivative (Fv 4-4-20) of the high affinity anti-fluorescein monoclonal antibody 4-4-20 complexed to anti-Met antibodies to study the influence of anti-Met antibodies of Fv 4-4-20 intervariable domain interactions. Anti-Met antibodies bound ... [truncated at 150 words]
Mallender WD, Carrero J, Voss EW Jr.
Comparative properties of the single chain antibody and Fv derivatives of mAb 4-4-20. Relationship between interdomain interactions and the high affinity for fluorescein ligand.
J Biol Chem. 1996; 271(10): 5338-46.Recombinant Fv derivative of the high affinity murine anti-fluorescein monoclonal antibody 4-4-20 was constructed and expressed in high yields, relative to the single chain antibody (SCA) derivative (2 3-fold), in Escherichia coli. Both variable heavy (VH) and variable light (VL) domains, that accumulated as insoluble inclusion bodies, were isolated, denatured, mixed, refolded, and affinity-purified to yield active Fv 4-4-20. Affinity-purified Fv 4-4-20 showed identical ligand binding properties compared with the SCA construct, both were slightly lower than the affinities expressed by Fab or IgG 4-4-20. Proper protein folding was shown to be domain-independent by in vitro mixing of individually refolded variable domains to yield functional Fv protein. In solid phase and solution phase assays, Fv 4-4-20 closely approximated the SCA derivative in terms of both idiotype and metatype, confirming identical active site structures and conformations. The equilibrium dissociation constant (Kd) for the VL/VH association (1.43 x 10(-7) M), which was ... [truncated at 150 words]
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Temperature and pH dependence of fluorescein binding within the monoclonal antibody 9-40 active site as monitored by hydrostatic pressure.
J Biol Chem. 1996; 271(10): 5332-7.In a comparative study, the thermodynamic parameter, DeltaV, was obtained using hydrostatic pressure-induced dissociation of fluorescein (Fl) from the active site of monoclonal antibody (mAb) 9-40 and its mutant and native derivatives equilibrated at six pH values (8.0, 7.5, 7.0, 6.5, 6.0, and 5.5) and four temperatures (35, 25, 15, and 5 degrees C). mAb 9-40 and its Fab and single-chain Fv (scFv) derivatives at pH 8.0 were found to have identical Fl dissociation behavior under pressure as a function of temperature. The pressure dissociation at 25 degrees C as a function of pH showed a sigmoidal dependence of DeltaV with a midpoint value at pH 7.4 for mAb 9-40. Comparison of experimental results for scFv 9-40/212 with its mutant scFv 9-40/212Arg-34L indicated that the pH dependence of mAb 9-40 was due to the titration of His-34L in the active site. Iodide quenching of bound Fl showed that the hapten ... [truncated at 150 words]
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Time-resolved fluorescence anisotropy of fluorescent-labeled lysophospholipid and taurodeoxycholate aggregates.
Biophys J. 1996; 70(3): 1466-71. PMCID: PMC1225073Previous work from this laboratory demonstrated that the environment-sensitive lysolipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- monomyristoylphosphatidylethanolamine (N-NBD-MPE), at concentrations below its critical micelle concentration (CMCN-NBD-MPE = 4 microM), reached maximum fluorescence yield upon the addition of taurodeoxycholate (TDC) at concentrations well below its CMC (CMCTDC = 2.5 mM). These data indicated the formation of micellar aggregates of the two amphiphiles at concentrations below both of their CMCs. In the present study, fluorescence lifetime and differential polarization measurements were made to determine the size of these aggregates. In the absence of TDC and at 0.5 mM TDC a single lifetime (tau) and rotational correlation time (phi) were measured for N-NBD-MPE at the submicellar concentration of 2 microM, indicating a lack of interaction between the two molecules at this concentration. Above 0.5 mM TDC, two discrete lifetimes were resolved. Based on these lifetimes, two distinct rotational correlation times were established through polarization measurements. The shorter phi(0.19-0.73 ... [truncated at 150 words]
Ehrhardt MR, Erijman L, Weber G, Wand AJ.
Molecular recognition by calmodulin: pressure-induced reorganization of a novel calmodulin-peptide complex.
Biochemistry. 1996; 35(5): 1599-1605.The interaction of apocalmodulin (apoCaM) with a peptide (Neurop) based on the primary sequence of the calmodulin-binding domain of neuromodulin has been studied by fluorescence spectroscopy. The 1:1 complex (12 microM) formed between apoCaM and the Neurop peptide is extremely sensitive to salt and is half dissociated in less than 0.1 M KCl, suggesting that electrostatic interactions contribute strongly to complex formation. Ion pair interactions are frequently sensitive to high hydrostatic pressure due to electrostriction effects in the solvated ion state. Application of high pressure to the apoCaM.Neurop complex causes a red shift of the Neurop tryptophan emission center of mass and a reduced residual anisotropy but with insignificant reduction in quantum yield. The transition is smooth, reversible, and apparently two-state with a midpoint pressure of approximately 0.8 kbar. The residual anisotropy, quantum yield, and center of mass of the emission spectrum are consistent with the movement of the tryptophan ... [truncated at 150 words]
Coelho-Sampaio T, Ferreira ST, Benaim G, Vieyra A.
Dissociation of purified erythrocyte Ca(2+)-ATPase by hydrostatic pressure.
J Biol Chem. 1991; 266(33): 22266-72.Subunit interactions in the Ca(2+)-ATPase from erythrocyte plasma membranes were investigated through a combination of fluorescence spectroscopy and high-pressure techniques. Application of hydrostatic pressure in the range of 1 bar to 2.4 kbar promoted full dissociation of the ATPase, as revealed by spectral shifts of the intrinsic fluorescence emission and by changes in the fluorescence polarization of dansyl-conjugated ATPase. Pressure dissociation of the ATPase displayed a dependence on protein concentration compatible with dissociation of a dimer. Calculated from pressure-dissociation curves, the standard volume change dV0 for the association of subunits was 43-50 ml/mol and K0, the dissociation constant at atmospheric pressure, was 6-9 x 10(-8) M. Addition of Ca2+ stabilized the dimeric ATPase structure against pressure dissociation, whereas addition of vanadate facilitated dissociation by pressure. These results suggest that intersubunit interactions depend on the equilibrium between the two major conformational states E1 and E2 of the ATPase. Addition of calmodulin ... [truncated at 150 words]
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Selective binding of cholesterol by recombinant fatty acid binding proteins.
J Biol Chem. 1991; 266(26): 17180-6.The sterol binding specificity of rat recombinant liver fatty acid binding protein (L-FABP) and intestinal fatty acid binding protein (I-FABP) was characterized with [3H]cholesterol and a fluorescent sterol analog dehydroergosterol. Ligand binding analysis, fluorescence spectroscopy, and activation of microsomal acyl-CoA:cholesterol acyltransferase activity showed that L-FABP-bound sterols. 1) Lipidex-1000 assay showed a dissociation constant Kd = 0.78 +/- 0.18 microM and stoichiometry of 0.47 +/- 0.16 mol/mol for [3H]cholesterol binding to L-PABP. 2) With [3H]cholesterol/phosphatidylcholine liposomes, the cholesterol binding parameters for L-FABP were Kd = 1.53 +/- 0.28 microM and stoichiometry 0.83 +/- 0.07 mol/mol. 3) L-FABP interaction with dehydroergosterol altered the fluorescence intensity and polarization of dehydroergosterol. Dehydroergosterol bound to L-FABP with Kd = 0.37 microM and a stoichiometry of 0.83 mol/mol. 4) Cholesterol and dehydroergosterol decreased L-FABP tyrosine lifetime. Dehydroergosterol binding produced sensitized emission of bound dehydroergosterol with longer lifetime.5) L-FABP bound two cis-parinaric acid molecules/molecule of protein. Cholesterol ... [truncated at 150 words]
Nemecz G, Jefferson JR, Schroeder F.
Polyene fatty acid interactions with recombinant intestinal and liver fatty acid-binding proteins. Spectroscopic studies.
J Biol Chem. 1991; 266(26): 17112-23.Binding and proximity relationships of fatty acids with recombinant rat liver fatty acid-binding protein (L-FABP) and intestinal fatty acid-binding protein (I-FABP) were studied with absorption and fluorescence spectroscopy. Protein aromatic amino acids were examined in the absence and presence of bound fatty acid. Second derivative absorbance spectroscopy of the apo- and holoproteins suggested that fatty acid binding altered the conformation of L-FABP, but not of I-FABP. Fatty acid binding also blocked the accessibility of L-FABP tyrosine and I-FABP tryptophan to Stern-Volmer quenching by acrylamide, indicating that these amino acids were present in the fatty acid-binding pocket. Forster energy transfer from I-FABP tryptophan to bound cis-parinaric acid resulted in quenching of tryptophan lifetime and appearance of sensitized lifetime of bound cis-parinaric acid. The calculated donor-acceptor distances were 16.9 +/- 0.6 and 19.2 +/- 0.3 A for I-FABP and L-FABP, respectively. Absorbance spectral shifts and ratios of fluorescence excitation maxima indicated that ... [truncated at 150 words]
Swindlehurst CA, Voss EW Jr.
Fluorescence measurements of immune complexes of Mab 4-4-20 with isomeric haptens.
Biophys J. 1991; 59(3): 619-28. PMCID: PMC1281226Relative differences in the active site environment of a monoclonal antibody when covalently bound to two isomeric haptens were studied using fluorescence quenching and lifetime measurements. Murine monoclonal antibody 4-4-20, a well-characterized high affinity antifluorescein antibody, served as the model IgG protein. Isomeric haptenic probes comparatively studied were fluorescein-5-isothiocyanate (FITC I, the immunogen) and fluorescein-6-isothiocyanate (FITC II). In kinetic binding studies, the association rate for the interaction of 4-4-20 with FITC I was greater than 2,000 times faster than the reaction with FITC II. Fluorescence lifetimes for FITC I covalently bound to 4-4-20 were 3.89 ns and 0.37 ns, indicative of hapten bound outside and inside the active site, respectively. Fluorescence lifetime for FITC II within the active site was indistinguishable from bound FITC I, indicating that interactions with active site residues which resulted in a decreased lifetime were similar for both isomers. A decreased lifetime for active site bound ... [truncated at 150 words]
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Structural microheterogeneity of a tryptophan residue required for efficient biological electron transfer between putidaredoxin and cytochrome P-450CAM.
Biochemistry. 1991; 30(7): 1845-51.The carboxy-terminal tryptophan of putidaredoxin, the Fe2S2.Cys4 iron-sulfur physiological redox partner of cytochrome P-450cam, is essential for maximal biological activity [Davies, M. D., Qin, L., Beck, J. L., Suslick, K. S., Koga, H., Horiuchi, T., & Sligar, S. G. (1990) J. Am. Chem. Soc. 112, 7396-7398]. This single tryptophan-containing protein thus represents an excellent system for studying the solution dynamics of a residue directly implicated in an electron-transfer pathway. Steady-state and time-resolved measurements of the tryptophan fluorescence have been conducted across the emission spectrum as a function of redox state to probe potential structural changes which might be candidates for structural gating phenomena. The steady-state emission spectrum (lambda max = 358 nm) and anisotropy (alpha = 0.04) suggest that Trp-106 is very solvent-exposed and rotating partially free of global protein constraints. The time-resolved fluorescence kinetics for both oxidized and reduced putidaredoxin are fit best with three discrete components of approximately ... [truncated at 150 words]
Wald JH, Krul ES, Jonas A.
Structure of apolipoprotein A-I in three homogeneous, reconstituted high density lipoprotein particles.
J Biol Chem. 1990; 265(32): 20037-43.To elucidate further the conformation of human apolipoprotein A-I (apoA-I) in lipid-bound states and its effect on the reaction with lecithin cholesterol acyltransferase (LCAT), we prepared reconstituted HDL (rHDL) particles from a reaction mixture containing dipalmitoylphosphatidylcholine/cholesterol/apoA-I in the molar ratios of 150:7.5:1. The particles were separated by gel filtration into three classes of highly homogeneous and reproducible discs with diameters of 97, 136, and 186 A, containing 2, 3, and 4 molecules of apoA-I/disc, respectively, and increasing proportions of phospholipid and cholesterol. These three classes of particles were then investigated by a variety of fluorescence techniques, to probe the average environment and mobility of the tryptophan (Trp) residues in the structure of apoA-I. We found small, gradual changes in the fluorescence parameters with changes in the size of the rHDL, consistent with a shift of Trp residues to a more hydrophobic and more rigid environment, as well as an increased ... [truncated at 150 words]
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Characteristics of self-quenching of the fluorescence of lipid-conjugated rhodamine in membranes.
J Biol Chem. 1990; 265(23): 13533-9.Self- or concentration quenching of octadecylrhodamine B (C18-Rh) fluorescence increases linearly in egg phosphatidylcholine (PC) vesicles but exponentially in vesicles composed of egg PC:cholesterol, 1:1, as the probe concentration is raised to 10 mol%. Cholesterol-dependent enhancement of self-quenching also occurs when N-(lissamine-rhodamine-B-sulfonyl)dioleoylphosphatidylethanolamine is substituted for C18-Rh and resembles that in dipalmitoylphosphatidylcholine vesicles below, as opposed to above, the phase transition. These effects are not due to changes in dimer:monomer absorbance. Stern-Volmer plots indicate a dependence of quenching on nonfluorescent dimers both in the presence and absence of cholesterol. Decreases in fluorescence lifetimes with increasing probe concentration parallel decreases in residual fluorescence of C18-Rh with increasing probe concentration in PC and PC + cholesterol membranes, respectively. Decreases in the steady-state polarization of C18-Rh fluorescence as its concentration is raised to 10 mol% indicate energy transfer with emission between probe molecules in PC and to a lesser extent in PC + cholesterol ... [truncated at 150 words]
Kao YL, Chong PLG, Huang CH.
Time-resolved fluorometric and differential scanning calorimetric investigation of dehydroergosterol in 1-stearoyl-2-caprylphosphatidylcholine bilayers.
Biochemistry. 1990; 29(5): 1315-22.Thermal and dynamic properties of dehydroergosterol (DHE) in 1-stearoyl-2-capryl-sn-glycero-3-phosphocholine [C(18):C(10)PC] have been studied by differential scanning calorimetry (DSC) and multifrequency phase-modulation fluorometry. C(18):C(10)PC is an asymmetric mixed-chain phosphatidylcholine known to form highly ordered mixed interdigitated bilayers below the maximal transition temperature, Tm, and partially interdigitated bilayers above Tm. This lipid system is thus unique in assessing the interactions between sterols and interdigitated lipid bilayers. DHE is a fluorescent analogue of cholesterol shown in previous studies to behave like cholesterol in noninterdigitated symmetric diacylphosphatidylcholines. DSC data show that DHE exhibits similar characteristics to cholesterol [Chong & Choate (1989) Biophys. J. 55, 551-556] in C(18):C(10)PC bilayers. DHE abolishes the phase transition of C(18):C(10)PC at 27 mol % compared to 25 mol % for cholesterol and decreases Tm, the onset temperature (To), and the completion temperature (Tc), at a similar rate to cholesterol at about -0.25 degrees C per mole percent DHE. Fluorescence ... [truncated at 150 words]
Dombrink-Kurtzman MA, Voss EW Jr.
Cryoprecipitation properties of a high-affinity monoclonal IgM anti-fluorescyl antibody.
Mol Immunol. 1988; 25(12): 1309-20.A high affinity (Ka = 2-3 x 10(10) M-1) murine monoclonal anti-fluorescein IgM antibody (18-2-3) exhibiting low temp. insolubility in the absence of bound ligand has served as a model to study cryoprecipitation. Insolubility of 18-2-3 at low temp. (4 degrees C) had been shown to be reversible at higher temp. and in the presence of fluorescyl ligand, indicating antigen binding site involvement. The primary objectives were to isolate and identify structural component(s) responsible for insolubility at low temp. Procedures developed for production and isolation of the monomeric subunit (IgMs) involved thiol reduction and gel filtration separation in the presence of the zwitterionic detergent, CHAPS. Fab and (Fc)5 fragments generated by papain digestion were purified sequentially by gel filtration (Sephadex G-200) and affinity chromatography (fluorescein-Sepharose). A protocol for covalent labeling of Fab fragments with the fluorescent chromophore 2,5 DNS-Cl was developed in order to measure steady-state fluorescence polarization. In kinetic ... [truncated at 150 words]
Stayton PS, Fisher MT, Sligar SG.
Determination of cytochrome b5 association reactions. Characterization of metmyoglobin and cytochrome P-450cam binding to genetically engineered cytochrome B5.
J Biol Chem. 1988; 263(27): 13544-8.Genetically engineered cytochrome b5 has been used to quantitative binding interactions of this protein with cytochrome P-450cam and sperm whale metmyoglobin by static fluorescence titration. Two cytochrome b5 mutants were constructed by cassette mutagenesis to replace a surface threonine residue with cysteine at two crystallographically defined positions, 65 and 8, located 11 and 21 A, respectively, from the nearest heme edge. The T65C and T8C mutant proteins were labeled with the sulfhydryl selective fluorescent reagent, acrylodan, which provided a spectral probe for monitoring protein-protein association. The fluorescence emission spectra of the acrylodan-labeled T65C mutant exhibited an ionic strength-dependent, blue-shifted fluorescence enhancement upon binding met-myoglobin, cytochrome c, and cytochrome P-450cam, whereas the acrylodan-labeled T8C mutant fluorescence emission remained unchanged during all titrations. Dissociation constants of 1.3, 0.6, and 0.5 microM, pH 7.15, were measured for metmyoglobin, cytochrome P-450cam, and cytochrome c, respectively. A similar averaged binding surface for cytochrome P-450cam and ... [truncated at 150 words]
