Resolution of mixtures of fluorophores using variable-frequency phase and modulation data

Gratton, Enrico; Limkeman, Mark; Lakowicz, Joseph R.; Maliwal, Badri P.; Cherek, Henryk; Laczko, Gabor

doi:10.1016/s0006-3495(84)84044-8

Cited by 262 publications

(162 citation statements)

References 8 publications

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“…In this later case the value of the phase can exceed 90 ° and the apparent phase lifetime can be negative. Phase and modulation data were analyzed using a non-linear least-squares routine described elsewhere [21,22] which fits the decay to a sum of exponentials.…”

Section: Resultsmentioning

confidence: 99%

Dynamic aspects of the heme-binding site in phylogenetically distant myoglobins

Bismuto¹,

Irace²,

Colonna³

et al. 1987

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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Section: Resultsmentioning

confidence: 99%

Dynamic aspects of the heme-binding site in phylogenetically distant myoglobins

Bismuto¹,

Irace²,

Colonna³

et al. 1987

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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“…Lifetime measurements were also done on the ISSK2 phase and modulation fluorometer (ISS Inc., Champaign, IL). Techniques used for phase fluorometry lifetime measurements and data analysis have been fully described previously (35)(36)(37)(38). The quality of fits was assessed by 2 values and by plots of weighed residuals.…”

Section: Methodsmentioning

confidence: 99%

Protein-RNA Interactions and Virus Stability as Probed by the Dynamics of Tryptophan Side Chains

Poian

Johnson

Silva

2002

Journal of Biological Chemistry

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The correlation between dynamics and stability of icosahedral viruses was studied by steady-state and timeresolved 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, timeresolved 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 of the x-ray structure. CPMV particles without RNA lost this discrete component of emission. Anisotropy decay measurements demonstrated that tryptophans rotate faster in empty particles when compared with the ribonucleoprotein particles. The increased structural breathing facilitates the denaturation of the empty particles. Our studies bring new insights into the intricate interactions between protein and RNA where part of the missing structural information on the nucleic acid molecule is compensated for by the dynamics.Virus assembly is a puzzle when viewed from the perspectives of thermodynamics and kinetics. Virus assembly touches questions related to protein folding, protein-protein interactions, and protein-nucleic acid interactions. As previously shown for several viruses, these questions are quite linked in many bacteria, plant, and animal viruses (1-5). Although much has been learned about the structure of many macromolecular assemblies, in most cases the direct correlation with function is not possible because of the lack of information about the dynamics of the system (4). In solution, icosahedral viruses can be viewed as molecular "quasicrystals," and they are good models for understanding the linkage among protein folding, protein-nucleic acid interactions, and macromolecular assembly.Comoviridae is a family of icosahedral plant viruses characterized by a divided genome. This genome consists in two single-stranded, positive-sense RNA molecules, which are encapsidated into distinct particles. The capsids contain equimolar amounts of a large (L) and a small (S) proteins. 1 The two RNA molecules are referred to as RNA 1 and RNA 2, with ϳ6.0 and 3.5 kb, respectively (6). Isolation of comovirus from infected plants results in a mixture of the two ribonucleoprotein particles, as well as empty shells. Thus, three different particles can be separated by gradient ultracentrifugation from preparations of these viruses: the top (no RNA), the middle (containing RNA 2), and the bottom (c...

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“…The methodology and data analysis have been fully described earlier [33][34][35]. For a single exponential decay one can readily obtain the lifetime from Eqns.…”

Section: Phase Fluorometry Lifetime Measurementsmentioning

confidence: 99%

“…A reasonable non-linear least-squares fit [33][34][35] [24] at 4°C for apparently similar RC complexes which, however, contained some unbond Chl. They found no dependence of the fluorescence decay parameters on the emission wavelength in the range from 670 to 690 nm.…”

Section: Analysis Of the Data: Discrete Component Analysismentioning

confidence: 99%

Chlorophyll a fluorescence lifetime distributions in open and closed Photosystem II reaction center preparations

Govindjee

Ven

Preston³

et al. 1990

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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We have measured the decay of chlorophyll a fluorescence at 4 °C under anaerobic conditions in stabilized photosystem !I reaction center complex isolated from spinach, using multifrequeucy (2-400 MHz) cross-correlation phase fluorometry. Examination of our data shows that although the fluorescence decay of open reaction centers (i.e., when both the electron donor P-680 and the electron acceptor pheophytin are capable of engaging in charge separation) can he analyzed as a multiexponential decay, another representation of the data is obtained when the decay is analyzed using a continuous distribution of lifetimes. [478][479][480][481][482][483][484][485][486] for the DI-D2-cytochrome /}-559 complex, obtained for F682 at 4°C by a time-resolved photon-counting spectrofiuorometer. When the reaction centers are closed by pretreatment with sodium dithionite and methyl viologen followed by exposure to laser excitation, conditions known to result in accumulation of reduced pheophytin, a dramatic decrease in the contribution of the slow lifetime component(s) is observed. These results suggest that the slow distribution lifetime component(s) in the 5-20 ns range originate(s) in the back reaction of the charge separated state. On the other hand, the fast lifetime component(s) in the picosecond range may he only partially related to the charge separation, since no dramatic change is observed upon closure of the reaction center. Perhaps, this component is related, in part, to the excitation energy migration among the various chromophores in the reaction center preparations.

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Resolution of mixtures of fluorophores using variable-frequency phase and modulation data

Cited by 262 publications

References 8 publications

Dynamic aspects of the heme-binding site in phylogenetically distant myoglobins

Dynamic aspects of the heme-binding site in phylogenetically distant myoglobins

Protein-RNA Interactions and Virus Stability as Probed by the Dynamics of Tryptophan Side Chains

Chlorophyll a fluorescence lifetime distributions in open and closed Photosystem II reaction center preparations

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