Abstract

Protein dynamics in cells have been shown to be markedly different from that in dilute solutions because of the highly crowded cellular interior. The volume exclusion arising from the high concentration of macromolecules present can affect both equilibrium and kinetic processes involving protein conformational changes. While global changes in structure leading to modulations in the stability of the protein have been well-documented, local changes that can have a large bearing on the functional aspects of these biomolecules are rare to come across. Using the multidomain serum protein human serum albumin and a fluorescence resonance energy transfer (FRET)-based approach, with fluorescent reporters in each of its three domains, we, in this article, have provided a detailed mapping of variations in the interdomain distances (as a function of pH) in the presence of five macromolecular crowding agents, differing based on their constituent monomers and average molecular weight(s). From the observation of correlated domain movements for dextran based crowding agents to anticorrelated motion induced by Ficoll 70, and both correlated and anticorrelated action for PEG8000 (PEG8), our results reveal the inherent complexity of a crowded milieu with the serum protein serving as an able sensor for decoding such variations. Differences in the manner in which the macromolecular crowders of similar average molecular weights influence the protein conformational ensemble also provide insights into the possible variations at the molecular level that these polymeric molecules possess. Evidence is presented in support of the fact that for the large molecular weight crowding agents and PEG8, soft interactions predominate over hard sphere potentials. Finally, the nature of domain movements encountered for the serum protein are of immense significance with respect to the function of human serum albumin (HSA) as a prolific binder and transporter of small molecules.

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