Study of protein folding under native conditions by rapidly switching the hydrostatic pressure inside an NMR sample cell

Significance Development of specialized instrumentation enables rapid switching of the hydrostatic pressure inside an operating NMR spectrometer. This technology allows observation of protein signals during the repeated folding process. Applied to ubiquitin, a previously extensively studied model of protein folding, the methodology reveals an initially highly dynamic state that deviates relatively little from random coil behavior but also provides evidence for numerous repeatedly failed folding events, previously only observed in computer simulations. Above room temperature, direct NMR evidence shows a ∼50% fraction of proteins folding through an on-pathway kinetic intermediate, thereby revealing two equally efficient parallel folding pathways.
In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. 15N and 13C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, 15N relaxation data show evidence for rapid exchange, on a ∼100-μs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ∼300 μM, oligomeric off-pathway intermediates compete with folding of the native state.