TRACKING NANOSECOND AND SUBNANOSECOND PROTEIN DYNAMICS ON-THE-FLY USING FREQUENCY-DOMAIN FLUORESCENCE

Fluorescence anisotropy and intensity decay experiments on proteins ca n provide detailed information on biomolecule dynamics and function. H owever, experiments of this sort are normally performed while the biom olecule is at or near equilibrium. Although information on protein dyn amics under equilibrium conditions is extremely important, details abo ut the protein behavior while it is actually undergoing change can pro vide significantly more insight into the overall protein behavior. Mul tiharmonic Fourier frequency-domain fluorescence provides a means to a cquire fluorescence anisotropy and intensity decay information on a re asonably rapid time scale. As a result, one can potentially track prot ein nanosecond and subnanosecond dynamical processes on-the-fly as the y undergo change(s) during, for example, protein-ligand binding, enzym atic reactions, or antigen/hapten-antibody association. To illustrate the potential of the frequency-domain on-the-fly methodology, we repor t here on the behavior of a model protein, bovine serum albumin, that has been labeled site-selectively with the fluorescent probe acrylodan (BSA-Ac). Conformational changes in the BSA-Ac are effected by using trypsin or beta-mercaptoethanol (BR-IE). BME is a disulfide interchang e reagent, and trypsin cleaves and excises from the entire BSA molecul e a 21 amino acid peptide segment that contains the covalently attache d Ac residue. This paper focuses on the time course of the fluorescenc e anisotropy and intensity decay kinetics of BSA-Ac as it reacts with trypsin or BME.