Residue Specific Resolution of Protein Folding Dynamics Using Isotope-Edited Infrared Temperature Jump Spectroscopy

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Abstract
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 13C18O isotope-edited infrared spectroscopy. A single 13C18O 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 α-helix of the protein. The 13C18O isotopic label shifted the carbonyl stretching frequency to 1572.1 cm-1 in the folded state, well removed from the 12C16O band of the unlabeled protein backbone. The unique IR signature of the 13C18O 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 12C16O band were found to be biphasic with similar relaxation rates. The slow relaxation phase (∼2 × 105 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 (∼107 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.