Excluded Volume and Weak Interactions in Crowded Solutions Modulate Conformations and RNA Binding of an Intrinsically Disordered Tail

The cellular milieu is a solution crowded with a significantconcentrationof different components (proteins, nucleic acids, metabolites, etc.). Such a crowded environment affects protein conformations,dynamics, and interactions. Intrinsically disordered proteins andregions are particularly sensitive to these effects. Here, we investigatethe impact on an intrinsically disordered tail that flanks a foldeddomain, the N-terminal domain, and the RNA-binding domain of the SARS-CoV-2nucleocapsid protein. We mimic the crowded environment of the cellusing polyethylene glycol (PEG) and study its impact on protein conformationsusing single-molecule Fo''rster resonance energy transfer. Wefound that high-molecular-weight PEG induces a collapse of the disorderedN-terminal tail, whereas low-molecular-weight PEG induces a chainexpansion. Our data can be explained by accounting for two opposingcontributions: favorable interactions between the protein and crowdermolecules and screening of excluded volume interactions. We furthercharacterized the interaction between protein and RNA in the presenceof crowding agents. While for all PEG molecules tested, we observedan increase in the binding affinity, the trend is not monotonic asa function of the degree of PEG polymerization. This points to therole of nonspecific protein-PEG interactions on binding inaddition to the entropic effects due to crowding. To separate theenthalpic and entropic components of the effects, we investigatedthe temperature dependence of the association constants in the absenceand presence of crowders. Finally, we compared the effects of crowdingacross mutations in the disordered region and found that the threefolddifference in association constants for two naturally occurring variantsof the SARS-CoV-2 nucleocapsid protein is reduced to almost identicalaffinities in the presence of crowders. Overall, our data providenew insights into understanding and modeling the contribution of crowdingeffects on disordered regions, including the impact of interactionsbetween proteins and crowders and their interplay when binding a ligand.

Automated summary

The crowded cellular environment has an impact on protein conformations, dynamics, and interactions, particularly on intrinsically disordered proteins and regions. This study investigates the effect of crowding using polyethylene glycol (PEG) on the intrinsically disordered tail of the SARS-CoV-2 nucleocapsid protein. It is found that high-molecular-weight PEG leads to the collapse of the disordered tail, while low-molecular-weight PEG causes chain expansion. The study also explores the interaction between the protein and RNA in the presence of crowding agents, revealing the role of nonspecific protein-PEG interactions and entropic effects.