Quantification of Entropic Excluded Volume Effects DrivingCrowding-Induced Collapse and Folding of a Disordered Protein

We investigate the conformational properties of the intrinsically disorderedDNA-binding domain of CytR in the presence of the polymeric crowder polyethylene glycol(PEG). Integrating circular dichroism, nuclear magnetic resonance, and single-moleculeFo''rster resonance energy transfer measurements, we demonstrate that disordered CytRpopulates a well-folded minor conformation in its native ensemble, while the unfoldedensemble collapses and folds with an increase in crowder density independent of thecrowder size. Employing a statistical-mechanical model, the effective reduction in theaccessible conformational space of a residue in the unfolded state is estimated to be 10% at300 mg/mL PEG8000, relative to dilute conditions. The experimentally consistent PEG-temperature phase diagram thusconstructed reveals that entropic effects can stabilize disordered CytR by 10 kJ mol-1, driving the equilibrium toward foldedconformations under physiological conditions. Our work highlights the malleable conformational landscape of CytR, the presence ofa folded conformation in the disordered ensemble, and proposes a scaling relation for quantifying excluded volume effects on proteinstability.

Automated summary

We investigated the conformational properties of the intrinsically disordered DNA-binding domain of CytR in the presence of polymeric crowder polyethylene glycol (PEG). Our findings demonstrate that the disordered CytR adopts a well-folded conformation in its native ensemble, while the unfolded ensemble collapses and folds with increasing crowder density, regardless of the size of the crowder. We estimate a 10% reduction in the accessible conformational space of a residue in the unfolded state at 300 mg/mL PEG8000. The experimentally constructed PEG-temperature phase diagram shows that entropic effects stabilize disordered CytR by 10 kJ mol-1, promoting the equilibrium towards folded conformations under physiological conditions. Our work highlights the flexible conformational landscape of CytR, reveals the presence of a folded conformation in the disordered ensemble, and proposes a scaling relation for quantifying excluded volume effects on protein stability.