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

AUTOR(ES)

Havlin, Robert H.

FONTE

National Academy of Sciences

RESUMO

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.

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