Simulation of fluorescence anisotropy experiments: Probing protein dynamics

¹ Theoretical and Computational Biophysics Department, Max-Planck-Institute for Biophysical Chemistry
² Department of Physics, Freie Universität

^* To whom correspondence should be addressed. E-mail: hgrubmu{at}gwdg.de.

Submitted on June 28, 2005
Revised on July 14, 2005
Accepted on 22 August 2005

	Abstract

Time-resolved fluorescence anisotropy decay experiments on a protein-attached dye can probe local protein dynamics and steric restrictions, but are difficult to interpret at the structural level. Aiming at an atomistic description, we have carried out molecular dynamics simulations of such experiments. Our simulations describe an Alexa488 fluorescent dye maleimide derivative covalently attached via a single cysteine to the AB-loop of bacteriorhodopsin. Fluorescence anisotropy decay curves obtained from the simulations agree well with the measured ones. Three anisotropy decay components were resolved and assigned 1) to the fast dynamics of the attached dye on the picosecond timescale, 2) to the slower dynamics of the loop at the one nanosecond timescale and 3) to the overall tumbling of the molecule. For the biologically relevant one nanosecond component we identified two processes from simulations, the motion of the flexible loop as well as slow conformational dynamics of the dye. These two processes are not separable by experiment alone. Furthermore, analysis of the correlation between the dye and the protein motion revealed which part and which motion of the protein is actually probed by the experiment. Finally, our simulations allowed to test the usual and inevitable assumption underlying these types of spectroscopic measurements that the attached dye probe does not severely perturb the protein dynamics. For the case at hand, by comparison with a simulation of the dye-free protein, the perturbation was quantified and found to be small.

Key Words: Molecular dynamics simulation, bacteriorhodopsin fragment, depolarization anisotropy spectroscopy, dye mobility, fluorescence spectroscopy, protein dynamics

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