1H NMR study of the effect of heme insertion on the folding of apomyoglobin

NMR signals arising from His EF5 and His GH1 N_ϵH protons of sperm whale myoglobin and apomyoglobin have been assigned, and the protein folding has been studied through the analysis of these signals. His EF5 and His GH1 N_ϵH protons participate in the internal hydrogen bonds at the B–GH and EF–H interfaces, respectively, and their signals are remarkably sensitive to local structural alterations at these sites. The shifts of these signals in alkaline pH condition were only slightly affected by the removal of heme, indicating that the overall protein folding is essentially retained in apoprotein. The line width of His EF5 proton signal, however, increased largely in the spectra of apomyoglobin and this result suggests a conformational lability of the EF–H interface in the absence of heme. Furthermore, the His EF5 proton signal was found to be influenced by not only the orientation of heme relative to the protein, but also by the type of hemin used to reconstitute apomyoglobin. These results clearly demonstrate the presence of a long-range structural correlation between the heme active site and the EF–H interface.