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Understanding of protein structure and stability gained to date has been acquired through investigations made under dilute conditions where total macromolecular concentration never surpasses 10 g l−1. However, biological macromolecules are known to evolve and function under crowded intracellular environments that comprises of proteins, nucleic acids, ribosomes and carbohydrates etc. Crowded environment is known to result in altered biological properties including thermodynamic, structural and functional aspect of macromolecules as compared to the macromolecules present in our commonly used experimental dilute buffers (for example, Tris HCl or phosphate buffer). In this study, we have investigated the thermodynamic and structural consequences of synthetic crowding agent (Ficoll 70) on three different proteins (Ribonuclease-A, lysozyme and holo α-lactalbumin) at different pH values. We report here that the effect of crowding is protein dependent in terms of protein thermal stability and structure. We also observed that the structural characteristics of the denatured state determines if crowding will have an effect or not on the protein stability.

The majority of in vitro investigations concerning protein aggregation have been performed in dilute systems, which poorly reflect the crowded in vivo scenario. Cell interior is highly crowded with soluble and insoluble macromolecules that alter macromolecular properties. Macromolecular crowding is known to enhance the rate and/or extent of protein aggregation. However, most of the understandings were derived from studies with α-rich or predominantly α-proteins. Indeed, α-proteins fold faster than β-proteins and conversion of α-helices to cross β-sheets are responsible for aggregate/amyloid formation. Therefore, it is important to investigate how macromolecular crowding affects the aggregation propensity of β-rich proteins. In this study, we investigated the effect of synthetic macromolecular crowders on bovine carbonic anhydrase (BCA, a β-rich protein) aggregation. In contrast to the effect of macromolecular crowding on α-rich proteins, BCA aggregation was observed to be reduced due to decrease in the population of aggregation-prone intermediates as a consequence of increased native state stability. In addition, the extent of aggregation was found to depend on the nature of the crowder under consideration. Combining the published data on α-proteins and this study, we conclude that macromolecular crowding can have opposite consequences on protein aggregation process depending on the fold type of the protein.

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