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

Abstract

The cellular milieu is a solution crowded with a significant concentration of different components (proteins, nucleic acids, metabolites, etc.). Such a crowded environment affects protein conformations, dynamics, and interactions. Intrinsically disordered proteins and regions are particularly sensitive to these effects. Here, we investigate the impact on an intrinsically disordered tail that flanks a folded domain, the N-terminal domain, and the RNA-binding domain of the SARS-CoV-2 nucleocapsid protein. We mimic the crowded environment of the cell using polyethylene glycol (PEG) and study its impact on protein conformations using single-molecule Forster resonance energy transfer. We found that high-molecular-weight PEG induces a collapse of the disordered N-terminal tail, whereas low-molecularweight PEG induces a chain expansion. Our data can be explained by accounting for two opposing contributions: favorable interactions between the protein and crowder molecules and screening of excluded volume interactions. We further characterized the interaction between protein and RNA in the presence of crowding agents. While for all PEG molecules tested, we observed an increase in the binding affinity, the trend is not monotonic as a function of the degree of PEG polymerization. This points to the role of nonspecific protein−PEG interactions on binding in addition to the entropic effects due to crowding. To separate the enthalpic and entropic components of the effects, we investigated the temperature dependence of the association constants in the absence and presence of crowders. Finally, we compared the effects of crowding across mutations in the disordered region and found that the threefold difference in association constants for two naturally occurring variants of the SARS-CoV-2 nucleocapsid protein is reduced to almost identical affinities in the presence of crowders. Overall, our data provide new insights into understanding and modeling the contribution of crowding effects on disordered regions, including the impact of interactions between proteins and crowders and their interplay when binding a ligand.

PEG-mCherry interactions beyond classical macromolecular crowding

“…Though PEG is often used as a mimetic for protein crowders because it is putatively inert, previous studies have reported attractive or repulsive PEG-protein interactions. 28,100 In this section, we investigate in detail the interactions between PEG and mCherry in our simulations. First, we computed the distribution of the number of PEG molecules that mCherry is in contact with throughout the simulations (Figure S10).…”

“…Previous studies have shown that crowding can lead to either expansion or compaction for IDPs. 7,28 However, IDPs most often experience compaction 9 since the excluded volume effect destabilizes extended conformational states. Several studies have reported that crowding can stabilize helical structures in IDPs, 7,9,20 which has also been attributed to the excluded volume effect.…”

“…Furthermore, relatively strong attractive interactions between PEG and protein have been observed using force experiments, which indicated that PEG is not an inert, simple polymeric crowder, but rather it can bind proteins. 15 Other studies 28,110 have also found specific interactions between PEG and proteins, suggesting that these interactions are likely a common feature of PEG-protein interactions.…”

“…20,27 Since soft interactions can be repulsive or attractive, 4,6,8 their influence on protein structure is varied, with some IDPs exhibiting compaction and others exhibiting expansion as a function of increasing crowder size or concentration. 7,28 For instance, non-monotonic changes in the radius of gyration with increasing crowder concentrations were observed for an IDP using small-angle neutron scattering. 7 The varied crowding-induced structural effects highlight the complexity of the relationship between protein structure and crowding and the challenge in predicting these effects.…”

Early-Career and Emerging Researchers in Physical Chemistry Volume 2

“…Understanding how macromolecules interact in the complex cellular environment is an important but difficult challenge because of crowding effects and weak interactions. Soranno and co-workers use single-molecule FRET under crowded conditions to measure the conformations of an intrinsically disordered protein tail that includes the RNA-binding domain of the SARS-CoV-2 nucleocapsid. They distinguish between the effects of excluded volume screening and weak interactions on the conformational dynamics and binding.…”