Pressure effect on the conformational fluctuation of apomyoglobin in the native state

We have investigated the effect of pressure on fluctuations of the native s tate 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 pre ssure range from 0.1 to 150 MPa at 293 K and pH 6.0. The H/D exchange of pr otons of the individual backbone amino acids in this pressure range was mon itored 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 tha n 100 compared to the values at 0.1 MPa. The amplitude of the decrease of t he protection factor varied among the individual amino acids on the same se condary structure unit. This result suggests that H/D exchange in apoMb is explained best by the penetration model, in which solvent penetrates into t he protein matrix via small motions. The result from limited proteolysis un der high pressure showed that a pressure increase does not induce local unf olding of the secondary structure units of apoMb. Conformational fluctuatio ns much smaller than local unfolding evidently provide pathways for water t o diffuse into the protein interior, and are enhanced by an increase of pre ssure.