Probing site-specific conformational distributions in protein folding with solid-state NMR.

We demonstrate an experimental approach to structural studies of unfolded and partially folded proteins in which conformational distributions are probed at a site-specific level by 2D solid-state 13C NMR spectroscopy of glassy frozen solutions. Experiments on chemical denaturation of the 35-residue villin headpiece subdomain, a model three-helix-bundle protein with a known folded structure, reveal that 13C-labeled residues in the three helical segments of the folded state have markedly different conformational distributions in the unfolded state. Moreover, the 2D solid-state NMR line shapes near the unfolding midpoint do not fit a simple two-state model, in which the conformational distributions of the unfolded component are assumed to be independent of denaturant concentration. Comparison with solid-state NMR spectra of peptides containing the individual helical segments suggests an alternative two-step description of conformational distributions in partially folded states of the helical villin headpiece subdomain, in which chemical denaturation is viewed as a disruption of tertiary contacts followed by equilibration of local secondary structure according to the intrinsic helical propensities of individual segments.