Using High Pressure NMR to Study Folding Cooperativity and Kinetics of Protein L9

Abstract

The ribosomal protein L9 is comprised of two globular domains, the N-terminal (NTL9) and C-terminal (CTL9) domains, connected by a rigid linker. The folding properties of the two domains have been investigated extensively with a variety of denaturants, including urea, GdnHCl, temperature, and pH. Both NTL9 and CTL9 have exhibited cooperative folding in a two-state manner. Pressure perturbation, as a relatively new denaturing factor, has been proved to unfold proteins locally by targeting their packing defects, and to slow protein folding due to the positive activation volume of folding. Conventional NMR spectroscopy is too slow to characterize the folding transition state of most single domain proteins. When combined with NMR spectroscopy, which provides residue-specific resolution, high pressure (HP) could reveal sequence based information regarding protein folding cooperativity and conformational dynamics. Hence we applied HP-NMR to NTL9, CTL9 and their variants, seeking to gain more insights on their folding mechanism. To study their folding equilibria, 1H-15N HSQC spectra were recorded as a function of pressure, and the intensity of each amide proton resonance was analyzed as a function of pressure. We combined HP with 15N ZZ-exchange NMR experiments to determine residue-specific folding and unfolding rate constants. Our thermodynamic and kinetic results reveal that NTL9 folding is very close to two-state, while small deviations from two-state behavior were observed for CTL9. The volumetric properties of these domains indicate that most of the solvent excluded voids in the hydrophobic cores of the folded structures are formed in their respective transition states.