Orientational averaging of dye molecules attached to proteinsin Forster resonance energy transfer measurements:Insights from a simulation study

摘要

Forster resonance energy transfer is an increasingly popular method for studying protein folding atsingle molecule resolution. By attaching dye molecules to particular residues in a protein moleculeand measuring the energy transfer to the acceptor dye on excitation of the donor dye, informationabout the separation of the dyes can be obtained. Here we use an atomistic coarse-grained molecularmodel of the protein and dyes to look at the assumption that the dyes rotate freely during the donordecay time. We find that although complete orientational averaging does not always occur, theconsequences of this are not extreme. Even in the native state, the errors in efficiency, which resultfrom incorrectly assuming k~2,- 2 / 3, are smaller than the typical experimental error of an efficiencymeasurement. The orientational freedom of the dyes originates both from the dynamics of the linkerand dye molecules and also from the movements of the protein chain itself. In the unfolded state, themovements of the protein chain are sufficient to provide complete, or almost complete, orientationalaveraging within the donor lifetime. Increasing the rigidity of the dyes therefore has only a verysmall effect on the measured efficiencies in the unfolded state. In the native state the contributionof the linker and dye dynamics to orientational averaging is larger; nevertheless increasing therigidity still has only a small effect on the measured efficiency.