Protein structural changes characterized by high-pressure, pulsed field gradient diffusion NMR spectroscopy

Abstract

Pulsed-field gradient NMR spectroscopy is widely used to measure the translational diffusion and hydrodynamic radius (Rh) of biomolecules in solution. For unfolded proteins, the Rh provides a sensitive reporter on the ensemble-averaged conformation and the extent of polypeptide chain expansion as a function of added denaturant. Hydrostatic pressure is a convenient and reversible alternative to chemical denaturants for the study of protein folding, and enables NMR measurements to be performed on a single sample. While the impact of pressure on the viscosity of water is well known, and our water diffusivity measurements agree closely with theoretical expectations, we find that elevated pressures increase the Rh of dioxane and other small molecules by amounts that correlate with their hydrophobicity, with parallel increases in rotational friction indicated by 13C longitudinal relaxation times. These data point to a tighter coupling with water for hydrophobic surfaces at elevated pressures. Translational diffusion measurement of the unfolded state of a pressure-sensitized ubiquitin mutant (VA2-ubiquitin) as a function of hydrostatic pressure or urea concentration shows that Rh values of both the folded and the unfolded states remain nearly invariant. At ca 23 Å, the Rh of the fully pressure-denatured state is essentially indistinguishable from the urea-denatured state, and close to the value expected for an idealized random coil of 76 residues. The intrinsically disordered protein (IDP) α-synuclein shows slight compaction at pressures above 2 kbar. Diffusion of unfolded ubiquitin and α-synuclein is significantly impacted by sample concentration, indicating that quantitative measurements need to be carried out under dilute conditions.