NIH Research Festival
FARE Award Winner
Protein folding is a complicated structural conversion process which lies at the heart of many biophysical and biological systems of interest. Time-resolved solid-state nuclear magnetic resonance (ssNMR) probes structural conversion processes by first triggering, then taking "snapshots" of structural changes by rapidly freezing after a variable evolution delay. Here we use sub-millisecond negative temperature jumps to trigger folding of the 35-residue villin headpiece subdomain (HP35), an ultra-fast folding protein studied extensively and widely used as a folding benchmark. A specially constructed temperature-jump apparatus utilizing copper capillary tubes anchored to temperature-controlled copper plates first heats HP35 solutions 95 ¬∞C, causing unfolding, then rapidly cools solutions to 30 ¬∞C in 0.6 ms to trigger folding. After a variable evolution time at 30 ¬∞C, HP35 is frozen in ~0.1 ms in a -145 ¬∞C isopentane bath. Frozen ensembles are studied using low-temperature ssNMR enhanced with dynamic nuclear polarization (DNP). 1D and 2D ssNMR spectra acquired as a function of the variable evolution time show signals consistent with native secondary structure forming on the sub-millisecond timescale, as expected from previous studies, but crucially provide evidence that full structural order, including the alignment of native sidechain configurations, forms much slower through a millisecond structural annealing process. Time-resolved ssNMR triggered by rapid temperature jumps offers direct access to short-lived structures and complexes and is applicable to diverse biomolecular and biophysical systems, and provides quantitative, atomistic information necessary to characterize this previously unobserved annealing process.
Scientific Focus Area: Structural Biology
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