Crowding-induced stabilization and destabilization in a single protein.

Abstract

The protein concentration in cells can reach 300 g/L. These crowded conditions affect protein stability. Classic crowding theories predict entropically driven stabilization, which occurs via steric repulsion, but growing evidence shows a role for non-covalent chemical interactions. To aid our understanding of physiologically relevant crowding, we used NMR-detected ¹H-²H exchange to examine a simple, semi-reductionist system: protein self-crowding at the residue level using the widely studied model globular protein, GB1 (the B1 domain streptococcal protein G) at concentrations up to its solubility limit, 100 g/L. The surprising result is that self-crowding stabilizes some residues but destabilizes others, contradicting predictions. Two other observations are also contradictory. First, temperature-dependence data show that stabilization can arise enthalpically, not just entropically. Second, concentration-dependence data show destabilization often increases with increasing concentration. These results show a key role for chemical interactions. More specifically, self-crowding increases the free energy required to expose those residues that are only exposed upon complete unfolding, and stabilization of these globally unfolding residues increases with GB1 concentration, a result we attribute to repulsive chemical interactions between GB1 molecules. On the other hand, residues exposed upon local unfolding tend to be destabilized, with destabilization increasing with concentration, a result we attribute to attractive chemical interactions between GB1 molecules.