Publications by Enrico Gratton before he founded the Laboratory for Fluorescence Dynamics (LFD) in 1986. More recent publications are listed as part of the LFD's publications.
Gratton E, Alcala JR, Marriott GM.
Rotations of tryptophan residues in proteins.
617th Meeting, Dundee.
Biochem Soc Trans. 1986; 14(5): 835-838.A classic study of the application of dynamic fluorescence depolarization methods to the investigation of protein internal rotations was reported some years ago by Munro et al. (1979). The relative large width of the excitation pulse used in those studies limited the time resolution to about 0.1 nanoseconds. Sommer et al. (1985) later measured the anisotropy decay of the single tyrosine residue in lima bean trypsin inhibitor using a femtosecond laser and streak camera. More recently, Harris et al (1986) have utilized time-correlated single photon counting with the narrow pulses obtained from a mode-locked laser to determine the rotational correlation times of the single tryptophan residue in mutants of T4 lysozyme. On the other hand, the use of differential phase and modulation ratio fluorometry to measure rotational properties of intrinsic fluorophores in proteins has been well established in our laboratory (Alcala et al., 1985, Gratton et al., 1985, Jameson et ... [truncated at 150 words]
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The statistical time correlation approach to enzyme action: the role of hydration.
The Fluctuating Enzyme (Nonequilibrium Problems in the Physical Sciences & Biology). By GR Welch (Editors). John Wiley & Sons, New York, pp. 227-262, 1986. ISBN: 9780471888703Introduction: From 'Structure and Function' to 'Relevant Correlated Events'.
Gratton E, Alcala JR, Marriott GM, Prendergast FG.
Fluorescence studies of protein dynamics.
Computer analysis for life science, Okayama, Japan, July 9-12, 1985.
Computer analysis for life science: Progress and challenges in biological and synthetic polymer research: proceedings of the International Symposium on. By C Kawabata and AR Bishop (Editors). Ohmsha, pp. 1-11, 1986. ISBN: 9784274072611Gratton E, Alcala JR, Marriott GM.
Rotational motions of tryptophan and tyrosine residues in proteins.
International Symposium on Structure and Dynamics of NucIeic Acids, Proteins, and Membranes. Aug 31-Sep 5, 1986, Riva del Garda, Italy.
Structure and Dynamics of Nucleic Acids, Proteins, and Membranes. By E Clementi and S Chin (Editors). Plenum Press, New York, pp. 149-151, 1986. ISBN: 030642553XProteins are flexible structures; their flexibility is crucial for biological function....
Parasassi T, Conti F, Gratton E.
Fluorophores in a polar medium: time dependence of emission spectra detected by multifrequency phase and modulation fluorometry.
Cell Mol Biol. 1986; 32(1): 99-102.Multifrequency phase and modulation fluorometry can be used to investigate the dipolar relaxation of fluorescent molecules in solvents, protein and membranes. The frequency-domain measurements permit calculation of time-resolved spectra, the time-resolved decay and the time course of the emission maximum and width. Theoretical comparison between the pulse and the harmonic response methods for the investigation of excited state processes are presented. Considerations on the two-state model are given.
Parasassi T, Conti F, Gratton E.
Time-resolved fluorescence emission spectra of Laurdan in phospholipid vesicles by multifrequency phase and modulation fluorometry.
Cell Mol Biol. 1986; 32(1): 103-8.The first time-resolved fluorescence emission spectra of Laurdan in phospholipid vesicles are reported, at temperatures corresponding to the membrane liquid crystallin phase and during the phase transition. Results were obtained using frequency-domain fluorescence spectroscopy. Fluorescence phase shift and modulation data were obtained at modulation frequencies ranging from 2 to 120 MHz and at different wavelengths. Data were analyzed for a double exponential decay and the results were used to construct the time-resolved emission spectra. The influence of the phosopholipid phase transition on the steady-state spectra and on the time-resolved spectra of Laurdan was then discussed.
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Wideband acousto-optic modulator for frequency domain fluorometry.
30th Annual Meeting of the Biophysical Society, San Francisco, California, February 1986.
Biophys J. 1986; 49(2 Pt 2): 467a, W-Pos73.The new technique of multifrequency phase fluorometry allows for accurate analysis of fluorescent activity using frequency domain measurements. So far, techniques for wideband modulation of light have been restricted to the Pockel's cell or intrinsically modulated sources such as the mode-locked laser and synchrotron radiation. A Pockel's cell modulator requires both a very well collumated beam and very high voltages for efficient modulation. We have developed an acoustooptic modulator which can be used with either CW laser sources and highly collumated arc lamps. The novelty of this new modulation scheme is that quasi-continuous modulation frequencies can be produced from DC to above 40 MHz and also from 80 MHz up to 200 MHz. The modulation efficiency is about 50%. and the RF power required to drive the modulator is only a few watts. In this new design, two acoustic standing waves are generated simultaneously in the same medium and modulation ... [truncated at 150 words]
Marriott GM, Alcala JR, Gratton E.
Rotations of tryptophan residues in lysozyme.
30th Annual Meeting of the Biophysical Society, San Francisco, California, February 1986.
Biophys J. 1986; 49(2 Pt 2): 493a, W-Pos147.A multi-frequency phase and modulation fluorometer using the harmonic content of a mode-locked laser was used to determine the rotational motions of tryptophan in hen egg white lysozyme. A mode-locked argon-ion laser was used to synchronously pump a rhodamine B dye laser. The pulse train output was amplitude modulated using an acousto-optic modulator and then frequency doubled. The frequency was variable from a few kilohertz to 500 megahertz. Using the differential phase and modulation technique to measure fluorescence lifetime and anisotropy decay, we were able to determine rotational correlation times of a few tens of picoseconds. In a study of the temperature and viscosity dependence of tryptophan motion in lysozyme, which has two tryptophan residues responsible for about 90% of the fluorescence, we were able to determine at least two rotational motions. The first corresponds at 20°C in aqueous solution to rotation of the entire macromolecule having a rotational correlation ... [truncated at 150 words]
Jameson DM, Eocleston JF, Gratton E.
Dynamic and steady-state fluorescence properties of elongation factor Tu.
30th Annual Meeting of the Biophysical Society, San Francisco, California, February 1986.
Biophys J. 1986; 49(2 Pt 2): 59a, M-PM-E11.The intrinsic fluorescence properties of elongation factor Tu (EF-Tu) in its complexes with GDP, elongation factor Ts and aurodox have been investigated using both steady-state spectral and polarization measurements and time-domain (lifetime and dynamic polarization) techniques. Upon excitation at 280 nm, the emission spectra of EF-TGDP and EF-Tu'EF-Ts are dominated by the tyrosine contribution. Excitation at 300 nm, however, results in exclusive emission from the unique tryptophan residue of the Tu protein. Multifrequency phase and modulation measurements were carried out over a broad range of modulation frequencies utilizing a novel modelocked laser system with ultraviolet capabilities. The lifetime of the single tryptophan residue in the various EF-Tu complexes was heterogeneous and was analyzed using a newly developed distributional approach. The lifetime distribution pattern was best fit by a bimodal distribution and was sensitive to the nature of the complex. Steady-state and dynamic polarization measurements demonstrate that the tryptophan residue in ... [truncated at 150 words]
Alcala JR, Marriott GM, Gratton E, Prendergast FG.
Fluorescence lifetime distributions in single tryptophan proteins.
30th Annual Meeting of the Biophysical Society, San Francisco, California, February 1986.
Biophys J. 1986; 49(2 Pt 2): 108a, M-Pos126.The conventional analysis of the fluorescence decay curve consists of determining the number and relative amplitude of exponential components. Our studies suggest that for some proteins this is not the case and the resolution of the decay curve using two or more exponentials is an approximation. Using our high resolution technique based on frequency domain fluorometry we show that the decay is better described by a continuous distribution of decay times. We have analyzed the observed decay using different distributions of lifetime values. The Lorentzian distribution function better described the data. We have also used the superposition of two and three different Lorentzians to better account for the real shape of the lifetime distribution. Furthermore, using this approach it is possible to determine if a distribution is unimodal or multimodal. We have studied the evolution of the lifetime distribution over a temperature range from 4C to 33C for a series ... [truncated at 150 words]
Prendergast FG, Francois JM, Alcala JR, Gratton E, Gerday C.
Changes in tryptophan fluorescence of eel Troponin-C mediated by binding of Ca2+ and lanthanides.
30th Annual Meeting of the Biophysical Society, San Francisco, California, February 1986.
Biophys J. 1986; 49(2 Pt 2): 107a, M-Pos125.The aminoacid sequence of eel troponin-C (TnC) is homologous with TnC from rabbit skeletal muscle but has a tryptophan substituted for a phenylalanine residue at a single location, and is devoid of tyrosine residues. The absence of tyrosine reduces the ambiguity in interpretation of the fluorescence emission properties of tryptophan in this protein. The protein binds Ca2+ and lanthanide ions with high affinity. Metal ion binding causes a 20 nm blue shift in the tryptophan emission spectrum, a decrease in the apparent accessibility of the fluorophore to water soluble quenchers, a marked increase in fluorescence anisotropy and a change in the near UV CD spectrum which is attributable to the tryptophan residue. We have used multifrequency phase fluorometry (with a laser excitation source) to examine the dynamics of the tryptophan moiety in the presence and absence of Ca++ as determined by time resolved intensity and anisotropy decays. Supported by GM34847.
Glaser M, Fiorini R, Wang S, Valentino M, Gratton E.
DPH fluorescence lifetime distributions in multilanellar vesicles.
30th Annual Meeting of the Biophysical Society, San Francisco, California, February 1986.
Biophys J. 1986; 49(2 Pt 2): 307a, T-Pos173.The emission decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles was measured using multifrequency phase fluorometry. The fluorescence decay, which is conventionally described by a sum of two or three exponentials, was analyzed using a continuous distribution of lifetime values. This approach better reflects the heterogeneity of the DPH surroundings and also the rate of exchange of environs during the excited state lifetime. The form of the distribution which better described the decay was lorentzian. In our analysis we have used the sum of two lorentzians to better describe a possible asymmetry of the real distribution. In most cases a simple lifetime distribution accounts for at least 95% of the observed decay. The width as well as the center position of the distribution are related to the structural phase transition in DPPC and DMPC. At temperatures below the phase transition the lifetime distribution is centered at ... [truncated at 150 words]
Wang S, Glaser M, Gratton E.
Frequency domain fluorescence studies of rotational motions of DPH in multilamellar vesicles.
30th Annual Meeting of the Biophysical Society, San Francisco, California, February 1986.
Biophys J. 1986; 49(2 Pt 2): 307a, T-Pos174.The physical structure of multilamellar vesicles has been studied using the rotational behavior of the fluorescent probe 1,6,-diphenyl-1,3,5-hexatriene in dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine. The decay of the emission anisotropy was analyzed using two emitting species in correspondence with the observation of two distinct lifetime components in the same system. The major component had an average lifetime value of about 10 nsec below the phospholipid phase transition and it decreased to about 7 nsec above the transition. The second component had a lifetime of approximately 3 nsec independent of temperature and contributed about 5 percent of the total fluorescence intensity. The major emitting component showed restricted motion. The values of the time zero anisotropy (ro), time infinity anisotropy (r inf ), the rotational correlation time and the average aperture of the cone were determined in the temperature range from 10°C to 60°C. Increasing the temperature through the phospholipid phase transition resulted in ... [truncated at 150 words]
Frauenfelder H, Gratton E.
Protein dynamics and hydration.
Biomembranes Part O: Protons and Water: Structure and Translocation (Methods in Enzymology, Vol. 127). Academic Press, pp. 207-216, 1986. ISBN: 0121820270Proteins are dynamic and not static systems; their internal motion is crucial for their function. Water is essential for protein motions and consequently also for protein function. Beyond these general statements, a full understanding of the relation between motion and structure and between hydration and motions is not yet available, and most of the essential features of the role of water for biological action remain to be elucidated. In this chapter we outline the general aspects of protein states and protein motions and discuss how water may be involved. This chapter is not a comprehensive guide to dynamics and hydration, but a modest road map to further work. To be specific, we will explain concepts and processes by using simple examples, but we believe that the phenomena are general and occur in various disguises in most proteins and probably also in nucleic acids.
Protein dynamics has been treated in a number ... [truncated at 150 words]
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Multifrequency phase fluorometry using pulsed sources: theory and applications.
Spectroscopy. 1986; 1(6): 28-36.Time-resolved fluorescence spectroscopy provides a unique tool for investigation of the structure and dynamics of biological molecules. Tlme-resolved fluorescence also is widely used for analytical purposes to quantitate the number of emitting species in a multicomponent system. Two alternative approaches have been used to record and analyze the time course of the emission: time-domain methods are based on the single-photon counting technique, and frequency-domain methods, which are generally referred to as phase fluorometry. Recent developments in phase fluorometry now permit the analysis of complex decay processes...
Scott AC, Gratton E, Shyamsunder E, Careri G.
IR overtone spectrum of the vibrational soliton in crystalline acetanilide.
Phys Rev B. 1985; 32(8): 5551-5553.The self-trapping (soliton) theory which was recently developed to account for the anomalous amide-I band at 1650 cm-1 in crystalline acetanilide (a model system for protein) has been extended to predict the anharmonicity constant of the overtone spectrum. These infrared-active overtones which have been detected at 3250, 4803, and 6304 cm-1 yield an anharmonicity constant that is in good agreement with the theory.
Mugnier J, Valeur B, Gratton E.
Rate of intramolecular electronic energy transfer in coumarin bichromophoric molecules. An investigation by multifrequency phase-modulation fluorometry.
Chem Phys Lett. 1985; 119(2-3): 217-222.Dynamics of intramolecular electronic energy transfer in bichromophoric molecules, consisting of two coumarines linked by a variable number of methylene groups is investigated by multifrequency phase-modulation fluorometry. From observation of the acceptor delayed emission, information on the rate of transfer can be obtained.
Gratton E, Alcala JR, Marriott GM, Prendergast FG.
Fluorescence studies of protein dynamics.
Proceedings of the International Symposium on ... Forum 85, Okayama, Japan, July 9-12, 1985.
Computer Analysis for Life Science. Progress and challenges in biological and synthetic polymer research. By C Kawabata and AR Bishop (Editors). Ohmsha, pp. 1-11, 1985. ISBN: 9784274072611-
New approach to phase and modulation resolved spectra.
Anal Chem. 1985; 57(8): 1694-1697.Time domain fluorescence spectrometry offers a versatile and powerful approach to the analysis of heterogeneous emitting systems. In this paper we describe a new approach, based on software, to the acquisition of phase and modulation resolved spectra. Mixtures of fluorophores with different lifetimes can be analyzed in real time to give the individual excitation or emission spectra. Examples of two- and three component mixtures are given and comparisons are made with the commercially available hardware approach.
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Ultrafast dynamics in proteins and membranes by time resolved fluorescence spectroscopy.
Structure and Motion: Membranes, Nucleic Acids, and Proteins. By E Clementi, G Corongiu, MH Sarma, and AR Sarma (Editors). Adenine Press, New York, pp. 155-168, 1985. ISBN: 0940030128
Lakowicz JR, Cherek H, Maliwal BP, Gratton E.
Time-resolved fluorescence anisotropies of diphenylhexatriene and perylene in solvents and lipid bilayers obtained by multifrequency phase-modulation fluorometry.
Biochemistry. 1985; 24(2): 376-383.Time-resolved decays of fluorescence anisotropy were obtained from frequency-domain measurements of the phase angle difference between the parallel and perpendicular components of the polarized emission and the ratio of the modulated amplitudes. These data were measured at modulation frequencies ranging from 1 to 200 MHz. To demonstrate the general applicability of this method, we describe the resolution of both simple and complex decays of anisotropy. In particular, we resolved single, double, and triple exponential decays of anisotropy and the hindered rotational motions of fluorophores within lipid bilayers. The ease and rapidity with which these results were obtained indicate that frequency-domain measurements are both practical and reliable for the determination of complex decays of anisotropy.
Alcala JR, Gratton E, Jameson DM.
A multifrequency phase fluorometer using the harmonic content of a mode-locked laser.
Anal Instrum. 1985; 14(3&4): 225-250.We describe the construction and operation of a cross-correlation phase and modulation fluorometer which uses the harmonic content of a high repetition rate mode-locked laser as the excitation source.
A mode-locked argon ion Laser is used to synchronously pump a dye laser. The pulse train output from the dye laser is amplitude modulated by an acousto-optic modulator and then frequency doubled with an angle tuned Frequency doubler. With the particular dye utilized in these studies, the ultraviolet light obtained was continuously tunable over the range 280-310 nm. In the frequency
doinain the high repetition rate pulsed source gives a large series of equally spaced harmonic frequencies. The frequency spacing of the harmonics is determined by the repeition frequency of the
laser. Amplitude modulation of the pulse train permits variation of the frequency quasi-continuously from a few hertz to gigahertz . Use of cross-correlation techniques permits precise isolation of individual frequencies. The ... [truncated at 150 words]
Lakowicz JR, Maliwal BP, Gratton E.
Recent developments in frequency-domain fluorometry.
Anal Instrum. 1985; 14(3&4): 193-223.Phase-modulation fluorometry is the frequency-domain analogue of time-resolved fluorescence spectroscopy. During the past three years we witnessed the development of variable-frequency phasemodulation fluorometers with modulation frequencies from 1 to 220 MHz. These instruments provide impressive resolution of multi or non-exponential fluorescence decays. To introduce these instruments we describe their design and operational principles. To illustrate the obtainable resolution we present results for the resolution of two and three-component mixtures of fluorophores, the resolution of complex anisotropy decays from non-spherical molecules and the determination of time-resolved emission spectra in the presence of time-dependent spectral relaxation. At present, the resolution obtainable with the frequency-domain fluorometers appears to be at least equivalent to that obtained with pulsed mode-locked laser sources with single photon counting. These mode-locked sources can also be used for phase fluorometry, as described elsewhere in this volume by Gratton and co-workers.
Lakowicz JR, Cherek H, Maliwal BP, Gratton E.
Frequency-domain phase-modulation fluorometry: resolution of complex decays of fluorescence intensity and anisotropy.
J Lumin. 1984; 31&32(2): 699-702.We used frequency-domain fluorometry to resolve multi-exponential decays of fluorescence intensity and anisotropy. The samples are excited with intensity modulated light, at modulation frequencies ranging from 1 to 200 MHz, and we measure the phase-shifts and demodulation factors. The decay times of a three-component mixture were resolved, 1.2, 4.1 and 7.7 nsec, as well as the two anisotropy decay times of the anisotropic rotator perylene.
Parasassi T, Conti F, Glaser M, Gratton E.
Detection of phospholipid phase separation. A multifrequency phase fluorimetry study of 1,6-diphenyl-1,3,5-hexatriene fluorescence.
J Biol Chem. 1984; 259(22): 14011-7.Using multifrequency phase and modulation fluorometry and a nonlinear least-squares analysis of lifetime data, we were able to determine the complex decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in synthetic phospholipid bilayers. Our results showed a monoexponential decay of DPH in the pure isotropic solvents studied, over a wide temperature range, and a double-exponential decay of DPH in phospholipids, both above and below the transition. During the transition, and in mixed-phase phospholipids, a three-component analysis was successfully accomplished, and the pre-exponential factors of the two main components have been shown to be quantitatively representative of the gel and liquid-crystalline phases of the bilayer. The fractional intensity of the shorter lifetime component depends on the modalities of the sample preparation. The factors affecting this component are discussed. From the DPH fluorescence lifetime and from the anisotropy data in L-alpha-dimyristoyl-phosphatidylcholine/L-alpha-dipalmitoyl-phosphatidyl choline mixtures, a phase diagram was independently constructed. Conclusions about the sensitivity and the partition ... [truncated at 150 words]
Parasassi T, Conti F, Gratton E.
Study of heterogeneous emission of parinaric acid isomers using multifrequency phase fluorometry.
Biochemistry. 1984; 23(23): 5660-5664.The fluorescence lifetimes of parinaric acid (PnA) isomers have been measured in pure solvents and in synthetic phospholipid membranes over a wide temperature range. In pure solvents, dodecane, ethanol, and cyclohexanol, the emission is well described by three exponential components of approximately 40, 12, and 2 ns. The preexponential factor of the long-lifetime component is quite small, and this component is neglected in the present study. The preexponential factors of the shorter lifetime components are strongly temperature dependent. Both cis- and trans-PnA show an inversion of the fractional contribution of these two components in a narrow temperature range. The inversion temperature is lower for trans-PnA than for cis-PnA, both in ethanol andin dodecane. The different temperature behavior of the decay of the two PnA isomers can influence their behavior in phospholipids. In L-α-dimyristoylphosphatidylcholine (DMPC) and L-α-dipalmitoylphosphatidylcholine (DPPC) membranes, the emission is more complex, and more than three components are necessary ... [truncated at 150 words]
Marque J, Eisenstein L, Gratton E, Sturtevant JM, Hardy CJ.
Thermodynamic properties of purple membrane.
Biophys J. 1984; 46(5): 567-72. PMCID: PMC1435054We measured the density, expansivity, specific heat at constant pressure, and sound velocity of suspensions of purple membrane from Halobacterium halobium and their constituent buffers. From these quantities we calculated the apparent values for the density, expansivity, adiabatic compressibility, isothermal compressibility, specific heat at constant pressure, and specific heat at constant volume for the purple membrane. These results are discussed with respect to previously reported measurements on globular proteins and lipids. Our data suggest a simple additive model in which the protein and lipid molecules expand and compress independently of each other. However, this simple model seems to fail to describe the specific heat data. Our compressibility data suggest that bacteriorhodopsin in native purple membrane binds less water than many globular proteins in neutral aqueous solution, a finding consistent with the lipid surround of bacteriorhodopsin in purple membrane.
Gratton E, Limkeman MK, Lakowicz JR, Maliwal BP, Cherek H, Laczko G.
Resolution of mixtures of fluorophores using variable-frequency phase and modulation data.
Biophys J. 1984; 46(4): 479-86. PMCID: PMC1435020We measured fluorescence phase shift and modulation data for one-, two- and, three-component mixtures of fluorophores at modulation frequencies ranging from 1 to 140 MHz. These data were analyzed using the least-squares procedure described in the preceding paper (Lakowicz, J. R., G. Laczko, M. Cherek, E. Gratton, and M. Limkeman, 1984, Biophys. J., 46:463-477). Using data obtained at a single emission bandpass, the lifetimes and preexponential factors of two-component mixtures could be easily resolved if the lifetimes differed by a factor of 2. With currently available instrumental stability, three-component mixtures could be resolved when the overall range of decay times was 10-fold, (e.g., 1.3, 4.4, and 12 ns). Measurement of phase and modulation data at several emission wavelengths, where the ratio of the preexponential factors varied, enhanced our ability to resolve closely spaced two and three-component decays. Two-component mixtures could then be resolved if the lifetimes differed by 30% (4.4 ... [truncated at 150 words]
Lakowicz JR, Laczko G, Cherek H, Gratton E, Limkeman MK.
Analysis of fluorescence decay kinetics from variable-frequency phase shift and modulation data.
Biophys J. 1984; 46(4): 463-77. PMCID: PMC1435017Recently it has become possible to measure fluorescence phase-shift and modulation data over a wide range of modulation frequencies. In this paper we describe the analysis of these data by the method of nonlinear least squares to determine the values of the lifetimes and fractional intensities for a mixture of exponentially decaying fluorophores. Analyzing simulated data allowed us to determine those experimental factors that are most critical for successfully resolving the emissions from mixtures of fluorophores. The most critical factors are the accuracy of the experimental data, the relative difference of the individual decay times, and the inclusion of data measured at multiple emission wavelengths. After measuring at eight widely spaced modulation frequencies, additional measurements yielded only a modest increase in resolution. In particular, the uncertainty in the parameters decreased approximately as the reciprocal of the square root of the number of modulation frequencies. Our simulations showed that with presently ... [truncated at 150 words]
Careri G, Buontempo U, Galluzzi F, Scott AC, Gratton E, Shyamsunder E.
Spectroscopic evidence for Davydov-like solitons in acetanilide.
Phys Rev B. 1984; 30(8): 4689-4702.Detailed measurements of infrared absorption and Raman scattering on crystalline acetanilide [(CH3CONHC6H5)x] at low temperature show a new band close to the conventional amide I band. Equilibrium properties and spectroscopic data rule out explanations based on a conventional assignment, crystal defects, Fermi resonance, and upon frozen kinetics between two different subsystems. Thus we cannot account for this band using the concepts of conventional molecular spectroscopy, but a soliton model, similar to that proposed by Davydov for α-helix in protein, is in satisfactory agreement with the experimental data.
Conti F, Parasassi T, Rosato N, Sapora O, Gratton E.
Fluorescence studies using synchrotron radiation on normal and differentiated cells labeled with parinaric acids.
Biochim Biophys Acta. 1984; 805(1): 117-22.Changes in membrane properties during the differentiation process in K562 cells have been investigated. A decrease of lectin-induced agglutination has been detected. The agglutination assay revealed to be an early and sensitive test to monitor the induced differentiation of the K562 cells. Naturally occurring fluorescent fatty acids (cis- and trans-parinaric acids) and the recently developed multifrequency phase and modulation technique were used to study cell membrane properties. Changes in fluorescence lifetime and polarization are clearly associated with cell differentiation, suggesting the involvement of the cellular plasma membrane in the differentiation process.
Lakowicz JR, Gratton E, Cherek H, Maliwal BP, Laczko G.
Determination of time-resolved fluorescence emission spectra and anisotropies of a fluorophore-protein complex using frequency-domain phase-modulation fluorometry.
J Biol Chem. 1984; 259(17): 10967-72.We report the first time-resolved fluorescence emission spectra and time-resolved fluorescence anisotropies obtained using frequency-domain fluorescence spectroscopy. We examined the fluorophore p-2-toluidinyl-6-naphthalenesulfonic acid (TNS) in viscous solvents and bound to the heme site of apomyoglobin using multifrequency phase fluorometers. Fluorescence phase shift and modulation data were obtained at modulation frequencies ranging from 1 to 200 MHz. For time-resolved emission spectra, the impulse response for the decay of intensity at each emission wavelength was obtained from the frequency response of the sample at the same emission wavelength. The decays have negative pre-exponential factors, consistent with a time-dependent spectral shift to longer wavelengths. These multiexponential decays were used to construct the time-resolved emission spectra, which were found to be in good agreement with earlier spectra obtained from time-domain measurements. Additionally, time-resolved anisotropies were obtained from the frequency-dependent phase angle differences between the parallel and perpendicularly polarized components of the emission. The rotational ... [truncated at 150 words]
Lakowicz JR, Cherek H, Laczko G, Gratton E.
Time-resolved fluorescence emission spectra of labeled phospholipid vesicles, as observed using multi-frequency phasemodulation fluorometry.
Biochim Biophys Acta. 1984; 777(2): 183-193.Multi-frequency phase-modulation fluorometry was used to determine the time-resolved spectral parameters of two different fluorescent probes, 6-palmitoyl-2-[(2-trimethylammonium)ethyl]methylamino]naphthalene chloride (Patman) and 2-p-toluidinyl-6-naphthalenesulfonic acid (TNS) in lipid vesicles. The frequency-domain measurements permitted calculation of time-resolved emission spectra, the time-resolved decays f the emission center of gravity and of the emission spectral width. Nanosecond spectral relaxation was found using both probes, and these rates increased with temperature. The detailed time-domain information available using our method indicated that spectral relaxation of TNS is mainly a continuous process, whereas relaxation of Patman shows the characteristic features of a stepwise relaxation. Our results indicate that complex excited-state processes can be quantified using frequency-domain fluorometry.
Jameson DM, Gratton E, Weber G, Alpert B.
Oxygen distribution and migration within Mbdes Fe and Hbdes Fe: multifrequency phase and modulation fluorometry study.
Biophys J. 1984; 45(4): 795-803. PMCID: PMC1434899Quenching of the intensity and lifetime of porphyrin fluorescence from Mbdes Fe and Hbdes Fe (iron-free myoglobin and hemoglobin) by oxygen was investigated using a multifrequency cross-correlation phase fluorometer. The single exponential decay characteristic of porphyrin emission of Mbdes Fe and Hbdes Fe became doubly exponential upon application of oxygen pressure. The results were interpreted in terms of a general model of dynamic quenching of fluorescence in globular proteins. The model accounted for the rate k+ of acquisition of quencher by the protein, the exit rate k- of quencher from the protein, and the migration rate chi of quencher in the protein interior. The values of k+, k-, and chi were different for Mbdes Fe and Hbdes Fe. The addition of 40% sucrose, which increased the bulk viscosity sixfold, modified these rates. These results are discussed and compared with previous quenching studies on proteins. The significance of these results and ... [truncated at 150 words]
Gratton E, Jameson DM, Weber G, Alpert B.
A model of dynamic quenching of fluorescence in globular proteins.
Biophys J. 1984; 45(4): 789-94. PMCID: PMC1434901A model is presented for the quenching of a fluorophore in a protein interior. At low quencher concentration the quenching process is determined by the acquisition rate of quencher by the protein, the migration rate of quencher in the protein interior, and the exit rate of quencher from the protein. In cases where the fluorescence emission observed in the absence of quencher could be described by a single exponential decay, the presence of quencher led to doubly exponential decay times, and the aforementioned exit rates of the quencher could be determined from experimental data. At high quencher concentration, the processes became more complex, and the deterministic rate equations used at low quencher concentration had to be modified to take into account the Poisson distribution of quencher molecules throughout the protein ensemble and also by using a migration rate for quencher in the protein interior that is a function of the ... [truncated at 150 words]
Gratton E, Jameson DM, Rosato N, Weber G.
Multifrequency cross-correlation phase fluorometer using synchroton radiation.
Rev Sci Instrum. 1984; 55(4): 486-494.The construction and operation of a cross-correlation phase and modulation fluorometer using the synchrotron radiation facility at the ADONE–Frascati electron storage ring is described. In the frequency domain the high repetition rate pulsed source gives a large series of equally spaced harmonic frequencies. Use of cross-correlation techniques in conjunction with such a light source permits one to isolate one harmonic frequency from the adjacent frequencies with high precision. The cross-correlation frequency required for the analysis of the phase delay and modulation ratio is obtained using two coupled frequency synthesizers, one of which drives the radio-frequency cavity of the storage ring and the other which modulates the response of the photomultipliers used for the signal detection. The accuracy, reproducibility, and sensitivity of the instrumentation have been determined on a number of systems and are reported.
Gratton E, Jameson DM, Rosato N, Weber G.
Multifrequency cross-correlation phase fluorometry using synchrotron radiation.
29th Annual Meeting of the Biophysical Society, San Antonio, Texas, February 19-23, 1984.
Biophys J. 1984; 45(2): 321a, W-Pos58.The construction and operation of a cross-correlation phase and modulation fluorometer using the synchrotron radiation facility at the ADONE-Frascati electron storage ring is described. In the frequency domain the pulsed source gives a large series of equally spaced harmonic frequencies. Use of cross-correlation techniques in conjunction with a high repetition rate pulsed light source permits one to isolate one harmonic frequency from the adjacent frequencies with high precision. The cross-correlation frequency required for the analysis of the phase delay and modulation ratio is obtained using two coupled frequency synthesizers, one of which drives the radiofrequency cavity of the storage ring and the other which modulates the response of the photomultipliers used for the signal detection. The accuracy, reproducibility and sensitivity of the instrumentation have been determined experimentally. A study of measurement artifacts related to the color error of the photomultipliers has been carried out and no sensible color error was ... [truncated at 150 words]
Lakowicz JR, Gratton E, Laczko G, Cherek H, Limkeman MK.
Analysis of fluorescence decay kinetics from variable-frequency phase shift and modulation data.
29th Annual Meeting of the Biophysical Society, San Antonio, Texas, February 19-23, 1984.
Biophys J. 1984; 45(2): 34a, M-PM-B11.It has recently become possible to measure fluorescence phase shift and modulation data over a wide range of modulation frequencies. Fluorescence phase shift and modulation data were obtained for one, two and three component mixtures of fluorophores at modulation frequencies ranging from 1 to 140 MHz. These data were analyzed using a least-squares procedure to determine the values of the lifetimes and fractional intensities for a mixture of exponentially decaying fluorophores. Using the data obtained at a single emission bandpass, the lifetimes and pre-exponential factors of two-component mixtures were easily and reliably resolved if the lifetimes differed by a factor of two. With currently available instrumental stability, and single emission bandpass data, three-component mixtures could just be resolved if the overall range of decay times was ten-fold, (1.3, 4.4 and 12 nsec). Measurement of phase and modulation data at several emission wavelengths where the ratio of the pre-exponential factors varied ... [truncated at 150 words]
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Fluorescence depolarization study of hydration-induced protein nanosecond internal rotational mobility.
29th Annual Meeting of the Biophysical Society, San Antonio, Texas, February 19-23, 1984.
Biophys J. 1984; 45(2): 126a, M-Pos212.The effect of hydration on protein dynamics was investigated using steady state fluorescence depolarization on azurin, which has a single tryptophan residue well-buried in the hydrophobic interior. Azurin was embedded in a thin, solid, water-permeable polymer film. This inhibited whole-protein motion while allowing examination of internal degrees of rotational freedom in the protein as a function of film hydration. In the dry film at room temperature the tryptophan fluorescence emission showed the limiting polarization characteristic of low temperature solutions in which all motion is frozen. As the hydration was increased, the observed polarization value changed sharply at about 0.6 h (h = g H20/g film), indicating an increase in the rotational mobility of the tryptophan. The fully hydrated film had a polarization equal to that of an azurin solution measurement extrapolated to infinite viscosity where the rotation of the protein as a whole was hindered. We conclude that the protein ... [truncated at 150 words]
Parasassi T, Conti F, Rosato N, Sapora O, Gratton E.
Fluorescence studies using synchrotron radiation on normal and differentiated cells labeled with parinaric acids.
29th Annual Meeting of the Biophysical Society, San Antonio, Texas, February 19-23, 1984.
Biophys J. 1984; 45(2): 201a, T-Pos67.The fluorescence lifetime and polarization of fatty acids (cis- and trans-parinaric acids) have been used to study changes in membrane properties during the differentiation process in K562 cells. Fluorescence lifetimes have been measured using the multifrequency phase fluorometer at the ADONE facility at Frascati (Italy). The emission of cis- and trans-parinaric acid in K562 cells is doubly exponential. Resolution of the two lifetime values and the relative fractional intensities has been performed. The relative fraction of each exponential component as well as the lifetime values are different in normal and differentiated cells. Lifetime and polarization value changes are clearly associated with differentiation process indicating the involvement of the cellular membrane in the differentiation process. Also the decrease of lectin-induced agglutination has been revealed to be an early and sensitive test to monitor K562 cells induced differentiation. Our results suggest a structural rearrangement of the plasma membrane after differentiation, which we ... [truncated at 150 words]
Gratton E, Jameson DM, Hall RD.
Multifrequency phase and modulation fluorometry.
Annu Rev Biophys Bioeng. 1984; 13: 105-24.Introduction: Determination of characteristic fluorescence parameters i.e. spectral properties, quantum yields, polarizations, and lifetimes, is important in the study of excited states of atoms, molecules, and crystals and also for diagnostic purposes in the physical, chemical, biological, and medical sciences. The appeal of fluorescence methodologies lies in their intrinsic sensitivity and also in the characteristic times case of the emission process...
Jameson DM, Gratton E, Hall RD.
The measurement and analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry.
App Spec Rev. 1984; 20(1): 55-106.During the last few decades fluorescence spectroscopy has developed into an important and widely used technique in the physical, chemical, and biological sciences. The increasing sophistication of theory and methodology has permitted us to apply fluorescence techniques profitably to a variety of complex problems. A fundamental consideration in the spectroscopic analysis of any system is the degree of sample heterogeneity. We wish to know, for example, if the heterogeneity arises from a melange of fluorophores (this condition often reduces to the question of sample purity) as a consequence of solvent-fluorophore interactions or from the fluorophore per se. An exact quantitation of the nature and extent of the heterogeneity may be critical to our understanding of the origins of that phenomenon. These data have important implications whether the desired information concerns a compositional description for analytical purposes or whether the fluorophore is designed to probe the molecular environment.
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A continuously variable frequency cross-correlation phase fluorometer with picosecond resolution.
Biophys J. 1983; 44(3): 315-24. PMCID: PMC1434839A detailed description of the construction and performance of a variable frequency cross-correlation phase fluorometer is reported. The phase fluorometer operates over the frequency range 1-160 MHz with a maximum resolution of a few picoseconds. The effects of distortions introduced by the light modulator and the nonlinear dynode characteristic are discussed in terms of the harmonic content of the detected signal. A source of systematic errors due to nonhomogeneous modulation is also discussed with particular attention to the color effect of the photomultipliers. The application of the phase fluorometer to the measurement of very long and very short lifetimes is reported. Some application to the measurement of multiexponential decays is also illustrated.
Gavish B, Gratton E, Hardy CJ, Denis AS.
Differential sound velocity apparatus for the investigation of protein solutions.
Rev Sci Instrum. 1983; 54(12): 1756-1760.A differential method for the measurement of sound velocity in solutions is described. The instrument uses two identical acoustic resonators and a newly developed electronic measuring system. The sound velocity is determined with a relative accuracy of ~ 3x10–6 over the entire frequency range 0.5–10 MHz. The instrument has been used to determine the compressibility of protein solutions and to study their velocity dispersion.
Gratton E, Lopez-Delgado R.
Remarks on a novel method for fluorescence lifetimes measurements.
NATO Advanced Study Institute on Time-Resolved Fluorescence Spectroscopy in Biochemistry and Biology. St. Andrews, Scotland. March 16-24, 1980.
Time-Resolved Fluorescence Spectroscopy in Biochemistry and Biology (NATO ASI Series A, Vol. 69). By RB Cundall and RE Dale (Editors). Plenum Press, pp. 109-114, 1983. ISBN: 0306414767It has been shown that, because of the high harmonic content of the narrow light pulses from a synchrotron radiation source, the sample may be considered as simultaneously excited by a set of
modulation frequencies up into the GHz region. By measuring the phase-shift of the fluorescence signal with respect t o t h e excitation signal in the high frequency region, time resolution in the picosecond range can be achieved. Also, since the harmonic content of the excitation source provides a wide range of modulation frequencies, the resolution of multi-exponential decays is possible, at least in principle.
Careri G, Buontempo U, Carta F, Gratton E, Scott AC.
Infrared absorption in acetanilide by solitons.
Phys Rev Lett. 1983; 51(4): 304-307.The infrared spectrum of acetanilide shows a new band that is red shifted from the main amide-I maximum by about 15 cm-1, the intensity of which increases at low temperature. It is suggested that this band may arise from the creation of amide-I solitons that are similar (but not identical) to those proposed by Davydov for the alpha helix in proteins.
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Microprocessor-controlled photon-counting spectrofluorometer.
Rev Sci Instrum. 1983; 54(3): 294-299.The construction details and performance characteristics for a fluorescence spectrophotometer are described. The system utilizes proton counting for signal detection and an Apple II microcomputer for data acquisition and analysis. Data-acquisition techniques are given along with typical excitation, emission, and polarization spectra. Particular reference is made to application of the instrument to fluorescence studies of protein/polymer films.
Gavish B, Gratton E, Hardy CJ.
Adiabatic compressibility of globular proteins.
Proc Natl Acad Sci USA. 1983; 80(3): 750-4. PMCID: PMC393457The adiabatic compressibility of several globular proteins has been measured by using an ultrasonic technique in the frequency range 0.5 to 10 MHz. The contributions to the measured compressibility from the protein matrix and from surface processes involving ionization of side chains and solvation effects are discussed. The internal protein compressibility is very low, indicating the existence of "dynamic domains" which are tentatively assigned to secondary structure elements.
Marque J, Hardy CJ, Gratton E, Eisenstein L.
Thermal expansion and adiabatic compression of purple membrane.
28th Annual Meeting of the Biophysical Society, San Diego, California, February 13-16, 1983.
Biophys J. 1983; 41(2): 335a, T-PM-Pos71.From measurements of the thermal expansion coefficient a = l/V(3V/DT)p, the adiabatic compressibility 6 = -l/V(aV/3P)S, and the density p of concentrated buffered aqueous suspensions of purple membrane from Halobacterium halobium, we have calculated the apparent expansibility, compressibility, and density of the purple membrane. Our data, taken together with data from previous studies on globular proteins and lipids, support a simple model in which the protein and lipid molecules expand and compress independently of each other. In addition, our compressibility data suggest that bacteriorhodopsin in native purple membrane binds less water than many globular proteins in neutral aqueous solution, a finding consistent with the lipid surround of bacteriorhodopsin in purple membrane and the preponderance of hydrophobic residues in bacteriorhodopsin. This work was supported in part by grants from the National Institutes of Health PHS 2 RO1 GM18051 and the National Science Foundation PCM 82-09616.
Rupley JA, Gratton E, Careri G.
Water and globular proteins.
Trends Biochem Sci. 1983; 8(1): 18-22.Dynamic, thermodynamic and structural studies of the hydration of globular proteins indicate how the macromolecule-water interface can influence folding, enzymatic activity and other biological properties.
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Analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry.
New Directions in Molecular Luminescence ASTM STP 822. By D Eastwood (Editors). American Society of Testing and Materials, Philadelphia, pp. 67-81, 1983. The technique of multifrequency phase and modulation fluorometry may be applied to the measurement and analysis of heterogeneous fluorescence lifetimes. This paper addresses two approaches to the data analysis: (1) the exact analytical solution (Weber's algorithm) for determining the lifetimes of components and their relative contributions using modulation frequencies and (2) a least-squares method. The mathematical rationale for each approach is described and the effects of random or systematic errors on the results obtained with each method are discussed. The relative merits of each approach are considered in light of results on both real and simulated data.
