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Tracking Nanosecond and Subnanosecond Protein Dynamics On-the-Fly Using Frequency-Domain Fluorescence

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Abstract:

Fluorescence anisotropy and intensity decay experiments on proteins can provide detailed information on biomolecule dynamics and function. However, experiments of this sort are normally performed while the biomolecule is at or near equilibrium. Although information on protein dynamics under equilibrium conditions is extremely important, details about the protein behavior while it is actually undergoing change can provide significantly more insight into the overall protein behavior. Multiharmonic Fourier frequency-domain fluorescence provides a means to acquire fluorescence anisotropy and intensity decay information on a reasonably rapid time scale. As a result, one can potentially track protein nanosecond and subnanosecond dynamical processes on-the-fly as they undergo change(s) during, for example, protein-ligand binding, enzymatic reactions, or antigen/hapten-antibody association. To illustrate the potential of the frequency-domain on-the-fly methodology, we report 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 (BME). BME is a disulfide interchange reagent, and trypsin cleaves and excises from the entire BSA molecule a 21 amino acid peptide segment that contains the covalently attached Ac residue. This paper focuses on the time course of the fluorescence anisotropy and intensity decay kinetics of BSA-Ac as it reacts with trypsin or BME.

Keywords: FLUORESCENCE ON-THE-FLY FREQUENCY-DOMAIN FLUORESCENCE NANOSECOND AND SUBNANOSECOND FLUORESCENCE ANISOTROPY AND INTENSITY DECAY KINETICS BOVINE SERUM ALBUMIN ACRYLODAN BETA -MERCAPTOETHANOL TRYPSIN DIGESTION

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/0003702981944742

Publication date: July 1, 1998

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