The Equilibrium Unfolding Parameters of Horse and Sperm Whale Myoglobin

Abstract

The urea and guanidine hydrochloride (GuHCl) unfolding of sperm whale ferrimyoglobin and of horse ferrimyoglobin has been studied under equilibrium conditions at both neutral and acid pH. Using two spectral parameters (the Soret absorbance and the molar ellipticity at 222 nm) to monitor conformational equilibria, and various thermodynamic treatments of the resulting data, it was found that the conformational free energy of unfolding, ΔG0 (i.e. the value in the absence of denaturant obtained using the two-state transition model), differs by about 20% in these two proteins. In particular, ΔG0 = 13.6 and 11.0 Cal per mole for sperm whale ferrimyoglobin and horse ferrimyoglobin, respectively. Based on the crystal structure of sperm whale ferrimyoglobin, an analysis of the 19 amino acid differences in the two proteins indicates that sperm whale ferrimyoglobin has a net of two additional arginyl-aspartyl side chain interactions that cannot be realized in horse ferrimyoglobin. Also, sperm whale ferrimyoglobin is predicted to be approximately 0.8 Cal per mole more stable than horse ferrimyoglobin due to an internal residue and several surface contact residues of higher hydrophobicity than the corresponding groups in horse ferrimyoglobin. Thus, the experimental free energy difference of 2.6 Cal per mole in the two proteins can be qualitatively explained, although minor conformation differences in localized regions of the native structures may exist. The acid unfolding of the two proteins can be described reasonably well in terms of six abnormal histidyl groups in each protein. These groups titrate with apparent pK values of 5.9 in the unfolded proteins and 3.0 and 3.7, respectively, in native sperm whale ferrimyoglobin and horse ferrimyoglobin. Corrections for the electrostatic free energy of the protein give an intrinsic pK of 6.6 for these groups in the unfolded state. A slightly better fit to the acid unfolding data could be achieved by considering two other classes, each containing 6 partially masked residues. The ΔG0 obtained from acid unfolding was in good agreement with that found from urea and GuHCl unfolding at neutral pH. These results show that conservative mutations can have pronounced effects on the conformational stability of phylogenetically similar globular proteins. However, in spite of the significant difference in ΔG0 of these two myoglobins, it is known that the oxygenation free energy differs by only a few per cent. Thus, these data demonstrate that in the myoglobins the evolutionary constraints are such that little variation is permitted in the biological function, but that appreciable variation is allowed in parts of the primary structure with concomitant changes in stability and perhaps small changes in conformation.