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.
Havlin
Robert H
RH
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA. robertt@niddk.nih.gov
Tycko
Robert
R
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003080:Cold Temperature; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D012997:Solvents
D012997:Solvents
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