Behavior of Bovine Serum Albumin Molecules in Molecular Crowding Environments Investigated by Raman Spectroscopy.

Abstract

The behavior of proteins in crowded environments is dominated by protein-crowder interactions (the entropic/excluded volume effect) and protein-protein interactions (the soft chemical effect). The details of these interactions, however, are not fully understood. In this study, the behavior of bovine serum albumin (BSA) in crowded environments, including high protein concentrations and in the presence of another protein, was investigated by Raman spectroscopy. A detailed analysis of the water, Tyr, and Phe Raman bands revealed that the excluded volume effect with an increase in the protein concentration changed the local environment of hydrophobic residues. In contrast, no specific changes to the secondary structure were observed from the analysis of the concentration dependence of the amide I band. BSA was experimentally shown to adopt a more compact state in the presence of the crowding agent. Moreover, H-D exchange experiments of the amide I band revealed that the intramolecular hydrogen bonds of BSA were strengthened in the presence of the protein crowder. Thus, the Raman spectroscopy results have revealed the molecular behavior of proteins in crowded environments by extracting information about the excluded volume effect, soft chemical interactions, and the hydration effect.