Structural Heterogeneity and Quantitative FRET Efficiency Distributions of Polyprolines through a Hybrid Atomistic Simulation and Monte Carlo Approach

Förster Resonance Energy Transfer (FRET) experiments probe molecular distances via distance dependent energy transfer from an excited donor dye to an acceptor dye. Single molecule experiments not only probe average distances, but also distance distributions or even fluctuations, and thus provide a powerful tool to study biomolecular structure and dynamics. However, the measured energy transfer efficiency depends not only on the distance between the dyes, but also on their mutual orientation, which is typically inaccessible to experiments. Thus, assumptions on the orientation distributions and averages are usually made, limiting the accuracy of the distance distributions extracted from FRET experiments. Here, we demonstrate that by combining single molecule FRET experiments with the mutual dye orientation statistics obtained from Molecular Dynamics (MD) simulations, improved estimates of distances and distributions are obtained. From the simulated time-dependent mutual orientations, FRET efficiencies are calculated and the full statistics of individual photon absorption, energy transfer, and photon emission events is obtained from subsequent Monte Carlo (MC) simulations of the FRET kinetics. All recorded emission events are collected to bursts from which efficiency distributions are calculated in close resemblance to the actual FRET experiment, taking shot noise fully into account. Using polyproline chains with attached Alexa 488 and Alexa 594 dyes as a test system, we demonstrate the feasibility of this approach by direct comparison to experimental data. We identified cis-isomers and different static local environments as sources of the experimentally observed heterogeneity. Reconstructions of distance distributions from experimental data at different levels of theory demonstrate how the respective underlying assumptions and approximations affect the obtained accuracy. Our results show that dye fluctuations obtained from MD simulations, combined with MC single photon kinetics, provide a versatile tool to improve the accuracy of distance distributions that can be extracted from measured single molecule FRET efficiencies

SINGLE-MOLECULE DETECTION OF RHODAMINE-6G IN ETHANOLIC SOLUTIONS USING CONTINUOUS WAVE LASER EXCITATION

The ultimate in analytical sensitivity is the ability to detect analytes on a single-molecule level. Laser-induced fluorescence (LIF) detection of single molecules in solution is hampered by specular, Rayleigh, and Raman scattering that contribute significantly to the background. In order to observe individual fluorescent molecules as they transit the laser beam in the presence of large backgrounds, it is necessary to detect a large number of photons per molecule. One method to increase the number of photons per event is to increase the residence time of the molecule in the laser beam. However, with long residence times, photostability sets an upper limit on the number of times the molecule can be cycled between the ground and first excited singlet state. We have observed the passage of individual rhodamine 6G (R-6G) molecules in ethanol (EtOH). The use of EtOH as a solvent allows one to obtain nearly 2 orders of magnitude more photons per molecule than may be obtained in H2O. Observation of single molecular events of R-6G in EtOH is substantiated by autocorrelation analysis and from shifts in the histograms of the frequency of photoelectron counts. Results from Monte Carlo simulations also support our experimental results.X11101sciescopu