Abstract
Fluorescence anisotropy decay is a popular optical technique to study the structure, size, shape, and even functions of biomolecules. The method measures the time dependence of the depolarization of a fluorophore and is therefore sensitive to the changes in the rotational motion (e.g., aggregation and binding) or changes in the mobility of segments of biopolymers (such as the ones associated with tertiary structure changes). Fluorescence anisotropy decay often requires the use of fluorescent dyes that need to be covalently attached to the biomolecule. The location of the attachment on the biomolecule (e.g., a protein) and the linker used, affect the mobility of the dye and its anisotropy decay. With this study we have combined the experimental data with molecular dynamic simulations to offer a more correct interpretation of the fluorescence anisotropy decay of a popular fluorescent dye (Atto 390) attached to the N-terminus of Hen Egg White Lysozyme (HEWL). Our model showed how the use of relatively simple molecular dynamics computation to simulate the motion of the dye, provide a model to interpret the experimental fluorescence anisotropy decay that yields a better estimate of the hydrodynamic radius of HEWL. The improvement is provided by a more detailed description of the segmental motion of the dye attached to the protein.