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JOURNAL ARTICLE

Orientational averaging of dye molecules attached to proteins in Förster resonance energy transfer measurements: insights from a simulation study

Lucy R Allen, Emanuele Paci
Journal of Chemical Physics 2009 August 14, 131 (6): 065101
19691411
Förster resonance energy transfer is an increasingly popular method for studying protein folding at single molecule resolution. By attaching dye molecules to particular residues in a protein molecule and measuring the energy transfer to the acceptor dye on excitation of the donor dye, information about the separation of the dyes can be obtained. Here we use an atomistic coarse-grained molecular model of the protein and dyes to look at the assumption that the dyes rotate freely during the donor decay time. We find that although complete orientational averaging does not always occur, the consequences of this are not extreme. Even in the native state, the errors in efficiency, which result from incorrectly assuming kappa2=2/3, are smaller than the typical experimental error of an efficiency measurement. The orientational freedom of the dyes originates both from the dynamics of the linker and dye molecules and also from the movements of the protein chain itself. In the unfolded state, the movements of the protein chain are sufficient to provide complete, or almost complete, orientational averaging within the donor lifetime. Increasing the rigidity of the dyes therefore has only a very small effect on the measured efficiencies in the unfolded state. In the native state the contribution of the linker and dye dynamics to orientational averaging is larger; nevertheless increasing the rigidity still has only a small effect on the measured efficiency.

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