1H-NMR study of inter-segmental hydrogen bonds in sperm whale and horse apomyoglobins.

NMR signals for HisB5 N(delta)H and HisEF5 N(epsilon)H protons of sperm whale and horse apomyoglobins were assigned and compared with the corresponding signals of the holoproteins in terms of pH and temperature dependence behaviors of their shifts and line widths in order to gain insight into structural difference between the apoproteins and the holoproteins. Since these protons are involved in internal hydrogen bonds at the interfaces between the B helix and the GH corner and between the EF corner and the H helix, local structures of the interfaces in these proteins have been inferred from the analyses of these signals. A large difference in the line width of HisEF5 N(epsilon)H proton signal between the apoproteins and the holoproteins strongly suggested that a sizable structural alteration is induced in the EF-H interface by the removal of heme. However, the results for HisB5 N(delta)H proton resonance indicated the absence of a significant structural alteration in the B-GH interface by heme extraction. These results are consistent with the data obtained from mutation [Hughson, F. M. & Baldwin, R. L. (1989) Biochemistry 28, 4415-4422] and amide-proton-exchange kinetic [Hughson, F. M., Wright, P. E. & Baldwin, R. L. (1990) Science 249, 1544-1548] studies, which indicated that the A, B, G and H helices in apomyoglobin maintain the same packing as they do in holoprotein.