High pressure NMR study of a small protein, gurmarin.
The effect of pressure on the structure of gurmarin, a globular, 35-residue protein from Gymnema sylvestre, was studied in aqueous environment (95% 1H2O/5% 2H2O, pH 2.0) with an on-line variable pressure NMR system operating at 750 MHz. Two-dimensional TOCSY and NOESY spectra were measured as functions of pressure between 1 and 2000 bar at 40 degrees C. Practically all the proton signals of gurmarin underwent some shifts with pressure, showing that the entire protein structure responds to, and is altered by, pressure. Most amide protons showed different degrees of low field shifts with pressure, namely 0-0.2 ppm with an average of 0.051 ppm at 2000 bar, showing that they are involved in hydrogen bonding and that these hydrogen bonds are shortened by pressure by different degrees. The tendency was also confirmed that the chemical shifts of the amide protons exposed to the solvent (water) are more sensitive to pressure than those internally hydrogen bonded with carbonyls. The pressure-induced shifts of the H alpha signals of the residues in the beta-sheet showed a negative correlation with the 'folding' shifts (difference between the shift at 1 bar and that of a random coil), suggesting that the main-chain torsion angles of the beta-sheet are slightly altered by pressure. Significant pressure-induced shifts were also observed for the side-chain protons (but no larger than 10% of the 'folding' shifts), demonstrating that the tertiary structure of gurmarin is also affected by pressure. Finally, the linearity of the pressure-induced shifts suggest that the compressibility of gurmarin is invariant in the pressure range between 1 and 2000 bar.
Inoue
K
K
Division of Molecular Science, Graduate School of Science and Technology, Kobe University, Japan.
Yamada
H
H
Imoto
T
T
Akasaka
K
K
D000595:Amino Acid Sequence; D017666:Deuterium Oxide; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D018515:Plant Leaves; D010940:Plant Proteins / Q000737:chemistry*; D011312:Pressure; D017433:Protein Structure, Secondary; D014867:Water
D010940:Plant Proteins; D014867:Water; C075206:Gurmarin protein, Gymnema sylvestre; D017666:Deuterium Oxide