Simulation of fluorescence anisotropy experiments: probing protein dynamics

Area of Science:

• Biophysics
• Computational Biology
• Structural Biology

Background:

• Time-resolved fluorescence anisotropy decay experiments offer insights into protein dynamics.
• Interpreting these experiments at a structural level is challenging due to complex motion.
• Protein-attached fluorescent dyes are used to probe local environments and steric restrictions.

Purpose of the Study:

• To provide an atomistic description of protein dynamics probed by attached fluorescent dyes.
• To interpret experimental fluorescence anisotropy decay data using molecular dynamics simulations.
• To quantify the perturbation of protein dynamics by the fluorescent dye probe.

Main Methods:

• Molecular dynamics simulations of an Alexa488 maleimide derivative attached to bacteriorhodopsin's AB-loop.
• Calculation of fluorescence anisotropy decay curves from simulation trajectories.
• Comparison of simulated decay curves with experimental data.

Main Results:

• Simulated anisotropy decay curves closely matched experimental measurements.
• Three decay components were resolved: fast dye dynamics (ps), loop dynamics (ns), and molecular tumbling.
• The 1-ns component revealed coupled loop motion and slow dye conformational changes, inseparable experimentally.
• Simulations identified the specific protein motions and regions probed by the dye.
• Perturbation of protein dynamics by the dye was quantified and found to be minimal.

Conclusions:

• Molecular dynamics simulations are valuable for interpreting fluorescence anisotropy decay experiments.
• The study successfully assigned decay components to specific dynamic processes.
• The fluorescent dye probe minimally perturbs the native protein dynamics.