NMR Snapshots of a Fluctuating Protein Structure: Ubiquitin at 30 bar–3 kbar Original Research Article

Conformational fluctuation plays a key role in protein function, but we know little about the associated structural changes. Here we present a general method for elucidating, at the atomic level, a large-scale shape change of a protein molecule in solution undergoing conformational fluctuation. The method utilizes the intimate relationship between conformation and partial molar volume and determines three-dimensional structures of a protein at different pressures using variable pressure NMR technique, whereby NOE distance and torsion angle constraints are used to create average coordinates. Ubiquitin (pH 4.6 at 20 °C) was chosen as the first target, for which structures were determined at 30 bar and at 3 kbar, giving “NMR snapshots” of a fluctuating protein structure at atomic resolution. The result reveals that the helix swings in and out by >3 Å with a simultaneous reorientation of the C-terminal segment, providing an “open” conformer suitable for enzyme recognition. Spin relaxation analysis indicates that this fluctuation occurs in the ten microsecond time range with activation volumes −4.2(±3.2) and 18.5(±3.0) ml/mol for the “closed-to-open” and the “open-to-closed” transitions, respectively.