NMR snapshots of a fluctuating protein structure: ubiquitin at 30 bar-3 kbar.
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 degrees 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 angstroms 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.
Kitahara
Ryo
R
Structural and Molecular Biology Laboratory, RIKEN Harima Institute at Spring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan.
Yokoyama
Shigeyuki
S
Akasaka
Kazuyuki
K
D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D013997:Time Factors; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism
D025801:Ubiquitin