Pressure effect on the conformational fluctuation of apomyoglobin in the native state.
We have investigated the effect of pressure on fluctuations of the native state of sperm whale apomyoglobin (apoMb) by H/D exchange, fluorescence, and limited proteolysis. The results from intrinsic fluorescence showed that a large fraction of apoMb molecules is in the native conformation in the pressure range from 0.1 to 150 MPa at 293 K and pH 6.0. The H/D exchange of protons of the individual backbone amino acids in this pressure range was monitored by NMR. The rate of H/D exchange was enhanced at high pressure, with the protection factors for some residues decreasing by factors of more than 100 compared to the values at 0.1 MPa. The amplitude of the decrease of the protection factor varied among the individual amino acids on the same secondary structure unit. This result suggests that H/D exchange in apoMb is explained best by the penetration model, in which solvent penetrates into the protein matrix via small motions. The result from limited proteolysis under high pressure showed that a pressure increase does not induce local unfolding of the secondary structure units of apoMb. Conformational fluctuations much smaller than local unfolding evidently provide pathways for water to diffuse into the protein interior, and are enhanced by an increase of pressure.
Tanaka
N
N
Department of Polymer Science & Engineering and Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606-8585, Japan. tanaka@ipc.kit.ac.jp
Ikeda
C
C
Kanaori
K
K
Hiraga
K
K
Konno
T
T
Kunugi
S
S
D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D003903:Deuterium; D004058:Diffusion; D006868:Hydrolysis; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000378:metabolism; D011312:Pressure; D011487:Protein Conformation; D011522:Protons; D012996:Solutions; D013050:Spectrometry, Fluorescence; D020860:Subtilisin / Q000378:metabolism; D013816:Thermodynamics; D014907:Whales
D001059:Apoproteins; D009211:Myoglobin; D010446:Peptide Fragments; D011522:Protons; D012996:Solutions; C010857:apomyoglobin; D003903:Deuterium; D020860:Subtilisin