Single-molecule Forster resonance energy transfer (FRET) between fluorescent donor and acceptor labels attached to a protein or nucleic acid is widely used to probe intramolecular distances and study the structure, dynamics and function of macromolecules. In these experiments, a molecule is illuminated by a laser, and the donor fluorophore is excited. The donor can emit a photon or transfer the excitation to an acceptor which then can emit a photon of a different color. The rate of transfer depends on the interdye distance and this is why there is information about conformational dynamics. The output of these experiments is a sequence of photons with recorded colors and arrival times. When a single molecule is excited by a pulsed laser, it is also possible to detect the time interval between the laser pulse and the photon. This so-called delay time is related to the fluorescence lifetime of the donor fluorophore. The distances between fluorescence labels attached to a molecule fluctuate due to conformational dynamics on a wide range of time scales. Extracting information about the dynamics is particularly challenging when the fluctuations are as fast as the time between photons. During the last year we have been working on extending our previous work to three color FRET in which three dyes are attached to the protein if interest( one donor and two acceptor). These experiments contain contain more information( three instead of one distances) than the usual two color FRET but the theory required to analyze them is also more challenging. We are in the process of submitting papers in this area and the results they contain will be discussed in next year's report. In a typical single-molecule force spectroscopy experiment, the ends of the molecule ( protein or nucleic acid)of interest are connected by long polymer linkers to a pair of mesoscopic beads trapped in the focus of two laser beams. At constant force load, the total extension, i.e., the end-to-end distance of the molecule plus linkers, is measured as a function of time. In the simplest systems, the measured extension fluctuates about two values characteristic of folded and unfolded states, with occasional transitions between them. Last year developed a quantitative theory of force spectroscopy experiments that accounts for the effects of the mesoscopic pulling device on the apparent rates of conformational transitions. We shown that the correct molecular (un)folding rates can be recovered from such trajectories, with a small linker correction, as long as the characteristic time of the bead fluctuations is shorter than the residence time in the unfolded (folded) state. In this reporting period in a paper entitled Transition paths in single-molecule force spectroscopy we have shown that accurate measurements of the molecular transition path times require an even faster apparatus response. Transition paths, the trajectory segments in which the molecule actually (un)folds, are properly resolved only if the beads fluctuate more rapidly than the end-to-end distance of the molecule. Therefore, over a wide regime, the measured rates may be meaningful but not the transition path times. Analytic expressions for the measured mean transition path times are obtained for systems diffusing anisotropically on a two-dimensional free energy surface. The observed transition path times depend on the properties both of the molecule and of the pulling device. The important implication our work is that recent experimental measurements of the duration apparent transition paths had little to do with what the molecule of interest was doing. Rather they reflected how the instrument responded to sudden changes of the molecular extension resulting from the conformational change that occurs when a random coil folds.
| Cossio, Pilar; Hummer, Gerhard; Szabo, Attila (2018) Transition paths in single-molecule force spectroscopy. J Chem Phys 148:123309 |
| Chung, Hoi Sung; Meng, Fanjie; Kim, Jae-Yeol et al. (2017) Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET. Proc Natl Acad Sci U S A 114:E6812-E6821 |
| Berezhkovskii, Alexander M; Szabo, Attila; Rotbart, T et al. (2017) Dependence of the Enzymatic Velocity on the Substrate Dissociation Rate. J Phys Chem B 121:3437-3442 |
| Hummer, Gerhard; Szabo, Attila (2017) Dynamics of the Orientational Factor in Fluorescence Resonance Energy Transfer. J Phys Chem B 121:3331-3339 |
| Gopich, Irina V; Szabo, Attila (2016) Reversible Stochastically Gated Diffusion-Influenced Reactions. J Phys Chem B 120:8080-9 |
| Cossio, Pilar; Hummer, Gerhard; Szabo, Attila (2016) Kinetic Ductility and Force-Spike Resistance of Proteins from Single-Molecule Force Spectroscopy. Biophys J 111:832-840 |
| Chung, Hoi Sung; Louis, John M; Gopich, Irina V (2016) Analysis of Fluorescence Lifetime and Energy Transfer Efficiency in Single-Molecule Photon Trajectories of Fast-Folding Proteins. J Phys Chem B 120:680-99 |
| Berezhkovskii, Alexander M; Szabo, Attila (2016) Theory of Crowding Effects on Bimolecular Reaction Rates. J Phys Chem B 120:5998-6002 |
| Gopich, Irina V; Szabo, Attila (2016) Influence of diffusion on the kinetics of multisite phosphorylation. Protein Sci 25:244-54 |
| Cossio, Pilar; Hummer, Gerhard; Szabo, Attila (2015) On artifacts in single-molecule force spectroscopy. Proc Natl Acad Sci U S A 112:14248-53 |
Showing the most recent 10 out of 21 publications
