Journal
BIOCHEMISTRY
Volume 46, Issue 11, Pages 3279-3285Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi602372y
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Funding
- NIGMS NIH HHS [GM 53640] Funding Source: Medline
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A major difficulty in experimental studies of protein folding is the lack of nonperturbing, residue specific probes of folding. Here, we demonstrate the ability to resolve protein folding dynamics at the level of a single residue using (CO)-C-13-O-18 isotope-edited infrared spectroscopy. A single (CO)-C-13-O-18 isotopic label was incorporated into the backbone of the 36 residue, three-helix bundle villin headpiece subdomain (HP36). The label was placed in a solvent protected region of the second alpha-helix of the protein. The (CO)-C-13-O-18 isotopic label shifted the carbonyl stretching frequency to 1572.1 cm(-1) in the folded state, well removed from the (CO)-C-12-O-16 band of the unlabeled protein backbone. The unique IR signature of the (CO)-C-13-O-18 label was exploited to probe the equilibrium thermal unfolding transition using temperature-dependent FTIR spectroscopy. The folding/unfolding dynamics were monitored using temperature-jump (T-jump) IR spectroscopy. The equilibrium unfolding studies showed conformational changes suggestive of a loss of helical structure in helix 2 prior to the global unfolding of the protein. T-jump relaxation kinetics probing both the labeled site and the (CO)-C-12-O-16 band were found to be biphasic with similar relaxation rates. The slow relaxation phase (similar to 2 x 10(5) s(-1)) corresponds to the global folding transition. The location of the label, a buried position in helix 2, provides an important probe of the origin of the fast relaxation phase (similar to 10(7) s(-1)). This phase has significant amplitude for the labeled position even though it is well protected from solvent in the folded structure. The fast phase likely represents a rapid pre-equilibrium that involves solvent penetration around the label and possible partial unfolding of helix 2 prior to the global unfolding transition. This work represents the first experimental study of ultrafast folding dynamics with residue specific resolution.
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