Two folded conformers of ubiquitin revealed by high-pressure NMR

High-pressure N-15/H-1 two-dimensional NMR spectroscopy has been utilized t o study conformational fluctuation of a 76-residue protein ubiquitin at pH 4.5 at 20 degreesC. The on-line variable pressure cell technique is used in conjunction with a high field NMR spectrometer operating at 750 MHz for H- 1 in the pressure range between 30 and 3500 bar. Large, continuous and reve rsible pressure-induced H-1 and N-15 chemical shifts were observed for 68 b ackbone amide groups, including the 7.52 ppm N-15 shift of Val70 at 3500 ba r, indicating a large-scale conformational change of ubiquitin with pressur e. On the basis of the analysis of sigmoid-shaped pressure shifts, we concl ude that ubiquitin exists as an equilibrium mixture of two major folded con formers mutually converting at a rate exceeding similar to 10(4) s(-1) at 2 0 degreesC at 2000 bar. The second conformer exists at a population of simi lar to 15% (DeltaG(0) = 4.2 kJ/mol) and is characterized with a significant ly smaller partial molar volume (DeltaV(0) = -24 mL/mol) than that of the w ell-known basic native conformer. The analysis of H-1 and N-15 pressure shi fts of individual amide groups indicates that the second conformer has a lo osened core structure with weakened hydrogen bonds in the five-stranded bet a -sheet. Furthermore, hydrogen bonds of residues 67-72 belonging to beta ( 5) are substantially weakened or partially broken, giving increased freedom of motion for the C-terminal segment. The latter is confirmed by the signi ficant decrease in N-15{H-1} nuclear Overhauser effect for residues beyond 70 at high pressure. Since the C-terminal carboxyl group constitutes the re active site for producing a multiubiquitin structure, the finding of the se cond folded conformer with a substantially altered conformation and mobilit y in the C-terminal region will shed new light on the reaction mechanism of ubiquitin.