Photon Counting Histogram: One‐Photon Excitation

Huang, Bo; Perroud, Thomas D.; Zare, Richard N.

doi:10.1002/cphc.200400176

Cited by 79 publications

(127 citation statements)

References 31 publications

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“…9 for all pairs of directly connected states. volume (22). This sensitivity also limits the utility of Brownian dynamics simulations of the molecule's trajectory through the laser spot.…”

Section: Resultsmentioning

confidence: 99%

Theory of the energy transfer efficiency and fluorescence lifetime distribution in single-molecule FRET

Gopich

Szabó

2012

Proc. Natl. Acad. Sci. U.S.A.

157

158

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In single-molecule FRET experiments with pulsed lasers, not only the colors of the photons but also the fluorescence lifetimes can be monitored. Although these quantities appear to be random, they are modulated by conformational dynamics. In order to extract information about such dynamics, we develop the theory of the joint distribution of FRET efficiencies and fluorescence lifetimes determined from bins (or bursts) of photons. Our starting point is a rigorous formal expression for the distribution of the numbers of donor and acceptor photons and donor lifetimes in a bin that treats the influence of conformational dynamics on all timescales. This formula leads to an analytic result for a two-state system interconverting on a timescale slower than the interphoton time and to an efficient simulation algorithm for multistate dynamics. The shape of the joint distribution contains more information about conformational dynamics than the FRET efficiency histogram alone. In favorable cases, the connectivity of the underlying conformational states can be determined directly by simple inspection of the projection of the joint distribution on the efficiency-lifetime plane.connectivity of kinetic schemes | diffusion | recoloring | quenching S ingle-molecule FRET experiments provide information about both structure and dynamics and have led to insights in a variety of biological processes including protein and RNA folding, enzyme catalysis, and protein-protein interactions (1-4). Consider a molecule with attached donor and acceptor fluorescent dyes. The donor is excited by a train of laser pulses (blue arrow in Fig. 1A). The excited donor can decay radiatively or nonradiatively or the excitation can be transferred to the acceptor. The excited acceptor can decay nonradiatively or by emitting a photon. The time between laser pulses (on the order of tens of nanoseconds) is much longer than the lifetimes of the excited states. The output of such an experiment is shown schematically in Fig. 1B. For each photon one can determine its color, arrival time, and delay time, which is the time interval between the laser pulse and the detection of the photon. For the sake of simplicity, only the delay times of the donor photons are shown, but acceptor delay times can readily be considered. The average of the delay times over the entire photon trajectory is the fluorescence lifetime of the donor in the presence of the acceptor. This experiment contains information about structure and dynamics because the photon colors and delay times depend on the rate of energy transfer. This rate in turn depends on the distance between the dyes (as distance −6 ) and their relative orientation, and can fluctuate on a variety of timescales from picoseconds to seconds.Suppose that the experimental photon trajectory is divided into time bins (see Fig. 1B). The FRET efficiency in a bin, E, is defined as the ratio of the acceptor photon counts to the total number of photons in a bin. We define the donor fluorescence lifetime in a bin, τ, as the sum of all don...

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“…9 for all pairs of directly connected states. volume (22). This sensitivity also limits the utility of Brownian dynamics simulations of the molecule's trajectory through the laser spot.…”

Section: Resultsmentioning

confidence: 99%

Theory of the energy transfer efficiency and fluorescence lifetime distribution in single-molecule FRET

Gopich

Szabó

2012

Proc. Natl. Acad. Sci. U.S.A.

157

158

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“…This fluorophore is believed to be unquenched ( i Ϸ 2.7 cpbm), which makes the total brightness of this species Ϸ3.1 cpbm. Under the current experimental conditions, PCH cannot resolve two species when the brightnesses differ by only 15% (6,31). Following the same logic, a third species should appear when the unquenched fluorescent species is labeled by another fluorophore.…”

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confidence: 78%

“…The construction of the PCHs for a given bin time and the determination of the resulting PCH parameters were similar to methods as described (6). Using fluorescence correlation spectroscopy, we determined a triplet relaxation time of 8 s, a triplet fraction of 15%, and a diffusion time of 96 s for TMR-cytochrome c. We chose a bin time of 20 s so that triplet formation and diffusion did not influence the PCH parameters by Ͼ10% (7).…”

Section: Methodsmentioning

confidence: 99%

“…Using fluorescence correlation spectroscopy, we determined a triplet relaxation time of 8 s, a triplet fraction of 15%, and a diffusion time of 96 s for TMR-cytochrome c. We chose a bin time of 20 s so that triplet formation and diffusion did not influence the PCH parameters by Ͼ10% (7). All histograms were fitted with one, two, and three species by using a second-order correction for the PCH model (6). The best model was chosen based on the goodness of the fit and the proper value for the semiempirical parameter F ϭ 0.67 Ϯ 0.07, which characterizes the deviation of the observation volume from a 3D Gaussian volume (5).…”

Section: Methodsmentioning

confidence: 99%

“…If different brightnesses correspond to specific protein conformations, PCH can resolve the heterogeneity of conformational states with single-molecule sensitivity. Using the PCH model for one-photon excitation for confocal microscopy, we successfully resolved a mixture of fluorophores characterized by different brightnesses (5,6). We also studied the effect of timedependent processes (such as diffusion and triplet formation) on the PCH parameters and determined optimal experimental conditions to minimize the influence of these processes (7).…”

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confidence: 99%

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Cytochrome c conformations resolved by the photon counting histogram: Watching the alkaline transition with single-molecule sensitivity

Perroud

Bokoch

Zare

2005

Proc. Natl. Acad. Sci. U.S.A.

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We apply the photon counting histogram (PCH) model, a fluorescence technique with single-molecule sensitivity, to study pHinduced conformational changes of cytochrome c. PCH is able to distinguish different protein conformations based on the brightness of a fluorophore sensitive to its local environment. We label cytochrome c through its single free cysteine with tetramethylrhodamine-5-maleimide (TMR), a fluorophore with specific brightnesses that we associate with specific protein conformations. Ensemble measurements demonstrate two different fluorescence responses with increasing pH: (i) a decrease in fluorescence intensity caused by the alkaline transition of cytochrome c (pH 7.0 -9.5), and (ii) an increase in intensity when the protein unfolds (pH 9.5-10.8). The magnitudes of these two responses depend strongly on the molar ratio of TMR used to label cytochrome c. Using PCH we determine that this effect arises from the proportion of a nonfunctional conformation in the sample, which can be differentiated from the functional conformation. We further determine the causes of each ensemble fluorescence response: (i) during the alkaline transition, the fluorophore enters a dark state and discrete conformations are observed, and (ii) as cytochrome c unfolds, the fluorophore incrementally brightens, but discrete conformations are no longer resolved. Moreover, we also show that functional TMR-cytochrome c undergoes a response of identical magnitude regardless of the proportion of nonfunctional protein in the sample. As expected for a technique with single-molecule sensitivity, we demonstrate that PCH can directly observe the most relevant conformation, unlike ensemble fluorometry.single-molecule fluorescence spectroscopy ͉ protein conformations ͉ protein labeling ͉ confocal microscopy ͉ metalloprotein I n cellular signaling networks, rare conformations of proteins are believed to be crucial for determining outcomes of the entire system (1, 2). Traditional biophysical methods rely on ensemble averages, that is, measurements from a large number of molecules simultaneously. As a consequence, rare, but important, populations can be obscured and go undetected. Recent advances in singlemolecule methodology have allowed researchers to break the ensemble average and more thoroughly characterize the heterogeneity of biological samples (3). This report describes the application of the photon counting histogram (PCH) model (4-7), a fluorescence spectroscopy technique with single-molecule sensitivity, to study the conformational heterogeneity of cytochrome c as a function of pH.Fluorescence spectroscopy has long been an important methodology for studying the conformational states of proteins, which is commonly done either by measuring the intrinsic fluorescence of the protein (for example, tryptophan residues) or covalently attaching an external fluorophore to specific residues (8). The latter approach assumes that the fluorophore does not significantly perturb the protein under investigation and that the photophysical propertie...

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Theory of Single‐Molecule FRET Efficiency Histograms

Gopich

Szabó

2011

Advances in Chemical Physics

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Photon Counting Histogram: One‐Photon Excitation

Cited by 79 publications

References 31 publications

Theory of the energy transfer efficiency and fluorescence lifetime distribution in single-molecule FRET

Theory of the energy transfer efficiency and fluorescence lifetime distribution in single-molecule FRET

Cytochrome c conformations resolved by the photon counting histogram: Watching the alkaline transition with single-molecule sensitivity

Theory of Single‐Molecule FRET Efficiency Histograms

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