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.