A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the conformational ensemble along the entire folding reaction coordinate. Simulations can provide this level of insight for small proteins. In contrast, with the exception of hydrogen exchange, which does not monitor folding directly, experimental studies of protein folding have not yielded such structural and energetic detail. NMR can provide residue specific atomic level structural information, but its implementation in protein folding studies using chemical or temperature perturbation is problematic. Here we present a highly detailed structural and energetic map of the entire folding landscape of the leucine-rich repeat protein, pp32 (Anp32), obtained by combining pressure-dependent site-specific H-N HSQC data with coarse-grained molecular dynamics simulations. The results obtained using this equilibrium approach demonstrate that the main barrier to folding of pp32 is quite broad and lies near the unfolded state, with structure apparent only in the C-terminal region. Significant deviation from two-state unfolding under pressure reveals an intermediate on the folded side of the main barrier in which the N-terminal region is disordered. A nonlinear temperature dependence of the population of this intermediate suggests a large heat capacity change associated with its formation. The combination of pressure, which favors the population of folding intermediates relative to chemical denaturants; NMR, which allows their observation; and constrained structure-based simulations yield unparalleled insight into protein folding mechanisms.
The effect of introducing internal cavities on protein native structure and global stability has been well documented, but the consequences of these packing defects on folding free-energy landscapes have received less attention. We investigated the effects of cavity creation on the folding landscape of the leucine-rich repeat protein pp32 by high-pressure (HP) and urea-dependent NMR and high-pressure small-angle X-ray scattering (HPSAXS). Despite a modest global energetic perturbation, cavity creation in the N-terminal capping motif (N-cap) resulted in very strong deviation from two-state unfolding behavior. In contrast, introduction of a cavity in the most stable, C-terminal half of pp32 led to highly concerted unfolding, presumably because the decrease in stability by the mutations attenuated the N- to C-terminal stability gradient present in WT pp32. Interestingly, enlarging the central cavity of the protein led to the population under pressure of a distinct intermediate in which the N-cap and repeats 1-4 were nearly completely unfolded, while the fifth repeat and the C-terminal capping motif remained fully folded. Thus, despite modest effects on global stability, introducing internal cavities can have starkly distinct repercussions on the conformational landscape of a protein, depending on their structural and energetic context.
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