Protein dynamics as a sensor for macromolecular crowding: Insights into mixed crowding

Understanding the effect of mixed crowding is of prime importance with regards to addressing the manner in which the crowded cellular interior influences the structure, function and dynamics of proteins. In this study we have used the internal dynamics of the multidomain serum protein, bovine serum albumin (BSA), labeled with the solvation probe 7-diethylamino-3-4-maleimidophenyl-4-methylcoumarin (CPM), as a sensor for binary crowding mixtures. Both synthetic (Ficoll 70, Dextran 70, Dextran 40 & PEG 8000) and protein based (unlabeled BSA) crowders have been used. The 'BSA + Ficoll 70' mixture had the maximum retardation effect on the protein dynamics with the average solvation time being more than that of the sum of the individual crowding agents. On the contrary, all other binary mixtures had the reverse effect with the excluded volume induced in their presence being lesser than that of the component crowders taken together. Further support was obtained from the overall protein dynamics based on the acrylamide quenching of emission of the tryptophan residues of BSA. Our results have been discussed in the light of the compatibility and lack in thereof, of the individual components of the mixtures, an aspect that has been further supported by excess viscosity (eta(E)) estimations based on our time resolved anisotropy data. Till date, crowding induced excluded volume has been sensed mostly through diffusion-based experiments or FRET based sensors. As shown in this study, internal protein dynamics can also add another dimension to the manner in which the degree of macromolecular congestion can be sensed, with the possibility of mapping site-specific response along the polypeptide backbone. (C) 2021 Elsevier B.V. All rights reserved.