Structural heterogeneity and quantitative FRET efficiency distributions of polyprolines through a hybrid atomistic simulation and Monte Carlo approach.

Helmut Grubmüller,Claus A M Seidel,Benjamin Schuler,Martin Hoefling,Nicola Lima,Jerome Mathe,Dominik Haenni

doi:10.1371/journal.pone.0019791

Abstract

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.

Highlights

Since the development of the Resonance Energy Transfer theory by Forster (FRET) in the late forties [1], and the definition of this technique as a ‘‘spectroscopic ruler’’ in biological systems by Stryer and Haugland [2], single molecule detection [3,4,5] and time-resolved experiments [6] have opened up a new window to probe inter- and intramolecular distances and motions
Because the isomerization times of minutes to hours are much longer than all other relevant time scales, we considered a weighted ensemble of all possible relevant isomerization states and performed the above Molecular Dynamics (MD) and Monte Carlo (MC) simulations with efficiency calculations separately for each isomer
We have demonstrated that structural information on the dynamics of Forster Resonance Energy Transfer (FRET) dye pairs from MD simulations improves the reconstruction of distances and distance distributions from experimental FRET efficiency distributions over the usual k2~2=3 approximation, which assumes isotropic and uncorrelated distributions of the dye transition dipole orientations

Summary

Introduction

Since the development of the Resonance Energy Transfer theory by Forster (FRET) in the late forties [1], and the definition of this technique as a ‘‘spectroscopic ruler’’ in biological systems by Stryer and Haugland [2], single molecule detection [3,4,5] and time-resolved experiments [6] have opened up a new window to probe inter- and intramolecular distances and motions. E depends on the distance R between the donor and the acceptor fluorophores, as well as on the mutual orientation of their respective transition dipole moments. R0 where R0 is the so-called Forster radius which denotes the distance at which 50% of the donor excitation is transferred to the acceptor molecule. This relation is widely used to monitor structural changes in biomolecules via FRET efficiency measurements [2,7]. Taking into account the flexibility of the fluorophores and their linkers, the measured intensities provide information on the mutual distance of these specific sites [8,9,10,11]. The use of multiple dye pairs allows for triangulation of biomolecules, which provides three-dimensional structural information [10,12,13,14,15,16]

Methods

Results

Conclusion