High-pressure 15N/1H two-dimensional NMR spectroscopy has been utilized to study conformational fluctuation of a 76-residue protein ubiquitin at pH 4.5 at 20 degrees C. The on-line variable pressure cell technique is used in conjunction with a high-field NMR spectrometer operating at 750 MHz for 1H in the pressure range between 30 and 3500 bar. Large, continuous and reversible pressure-induced 1H and 15N chemical shifts were observed for 68 backbone amide groups, including the 7.52 ppm 15N shift of Val70 at 3500 bar, indicating a large-scale conformational change of ubiquitin with pressure. On the basis of the analysis of sigmoid-shaped pressure shifts, we conclude that ubiquitin exists as an equilibrium mixture of two major folded conformers mutually converting at a rate exceeding approximately 10(4) s(-1) at 20 degrees C at 2000 bar. The second conformer exists at a population of approximately 15% (DeltaG(0) = 4.2 kJ/mol) and is characterized with a significantly smaller partial molar volume (DeltaV(0) = -24 mL/mol) than that of the well-known basic native conformer. The analysis of 1H and 15N pressure shifts of individual amide groups indicates that the second conformer has a loosened core structure with weakened hydrogen bonds in the five-stranded beta-sheet. Furthermore, hydrogen bonds of residues 67-72 belonging to beta5 are substantially weakened or partially broken, giving increased freedom of motion for the C-terminal segment. The latter is confirmed by the significant decrease in 15N[1H] nuclear Overhauser effect for residues beyond 70 at high pressure. Since the C-terminal carboxyl group constitutes the reactive site for producing a multi-ubiquitin structure, the finding of the second folded conformer with a substantially altered conformation and mobility in the C-terminal region will shed new light on the reaction mechanism of ubiquitin.