Studying protein folding cooperativity with pressure perturbation

Abstract

PP32 is a leucine rich repeat protein with capping motifs on both termini. We examined the role of the capping motifs in defining its folding cooperativity by investigating pressure-induced unfolding of an N-terminal capping domain (N-cap) deletion mutant and two C-terminal capping motif (C-cap) destabilization mutants, using high pressure NMR, SAXS, and fluorescence. We found that the N- and C-caps have distinct effects on the folding cooperativity of PP32, which arises from the differential free energy distribution in the

Furthermore, understanding sequence dependence of protein folding requires determination of residue-specific kinetic rates. We combined high pressure with ZZ-exchange spectroscopy, and measured the residue-specific rates for the N-terminal domain of L9 (NTL9) and a CTL9 mutant. Our results revealed that NTL9 folding is close to two-state, whereas small deviations from kinetic cooperativity were observed for CTL9, and that most of the solvent excluded voids in the hydrophobic cores of their native structures are already formed in the respective transition states. In conclusion, the work presented in this dissertation demonstrates pressure can indeed facilitate us to characterize key determinants encoded in the primary sequence of protein folding cooperativity.

protein. The C-terminal domain of the ribosomal protein L9 (CTL9) is a small globular protein. Pressure and temperature dependent NMR revealed significant deviations from two-state behavior for CTL9, with its hydrophobic core selectively destabilized by increasing temperature. Yet the I98A mutation in the core resulted in highly cooperative pressure unfolding. These observations indicate that local stability, as opposed to long-range interactions, determines folding cooperativity.

The simplest picture of protein folding envisages only two significantly populated states, native and unfolded, separated by a single free energy barrier, and interconverting in a cooperative two-state manner. Despite the large magnitude of interactions required for folding process, the simple two-state model has been successfully applied for many small single domain proteins. However, how the primary sequence determines the folding cooperativity remains a major challenge. Pressure, which acts mainly to eliminate local packing defects in the folded structure, can facilitate the observation of folding intermediates, and hence can provide exclusive insights into protein folding mechanisms. Here we applied pressure perturbation combined with NMR and other biophysical approaches on two small single domain proteins, PP32 and CTL9, to investigate the structural and energetic determinants of their folding cooperativity.