Structural Characterization of a Ubiquitin Folding Intermediate by Pressure-Jump NMR

Abstract

Small proteins rapidly fold on the timescale of milliseconds or less. Proteins with a substantial difference between the volumes of the folded and unfolded states experience large shifts in thermodynamic equilibrium upon variation of hydrostatic pressure, enabling experimental control over folding and unfolding. Using hardware that performs rapid and repeatable pressure switching within an NMR sample cell, we study the folding process of a pressure-sensitized mutant of ubiquitin in the absence of denaturants. This approach makes it possible to record 2D and 3D NMR spectra of the folding protein at atmospheric pressure, and to monitor chemical shift changes with sub-millisecond resolution, providing residue-specific information on the folding process. 1H, 15N, and 13C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. Combining time-resolved measurements of NMR observables, we develop a structural model for the folding intermediate, providing new insight into folding dynamics and kinetic traps present even in small, single-domain proteins.