Local Dynamics of FRET Dyes in an Intrinsically Disordered Protein Studied by MD Simulations

Abstract

Fluorescence resonance energy transfer (FRET) is widely used to track structural changes of biomolecules such as conformational transitions, aggregation or folding. The energy transfer efficiency between a donor-acceptor dye pair is a measure of their mutual separation.However, several assumptions about the dynamics of the dyes have to be made to quantitatively interpret the data measured in single molecule or bulk experiments. In particular, the mutual orientation of their transition dipole vectors determines the FRET efficiency, and has to be disentangled from the inter-dye distance. To this aim, it is usually assumed that both dyes adopt an isotropic orientation and that their orientational fluctuations are fast compared to their excitation decay rate. In this case, an orientation factor of κ2 = 2/3 is obtained.In extended molecular dynamics (MD) simulations we investigated the local flexibility of FRET pairs (tryptophan as donor, coumarin as acceptor) located in the N-terminal and NAC region of alpha-synuclein (aS) which is an entirely intrinsically disordered protein (IDP) of 14.4kD. MD simulations of FRET probes yield instantaneous information about their mutual orientation κ2 and distance RDA which are typically inaccessible to experiments.The orientation factors obtained from our MD simulations deviated considerably from the isotropic value κ2 = 2/3. Further, rotational autocorrelation functions obtained for both dyes reveal a slow diffusion on the surface of the protein. Unexpectedly, we found that slow internal structural fluctuations of aS can cause transient immobilization of one or both dyes, which markedly affects the interpretation of FRET data and might be a general feature of FRET probes attached to IDPs.M. Hoefling, N.Lima, D.Haenni, C.A. M.Seidel, B.Schuler, H.Grubmuller, PLoSONE 6(5): e19791. doi:10.1371/journal.pone.0019791.A.Grupi, E.Haas, JMB, 405:1267-1283 (2011).B.W. van der Meer, Rev.Mol.Biotechnology, 82:181-196 (2002).