PROBING THE CONFORMATIONAL STATE OF APOMYOGLOBIN BY LIMITED PROTEOLYSIS

We show here that limited proteolysis can probe the structural and dyn amic differences between the hole and apo form of horse myoglobin (Mb) . Initial nicking of the polypeptide chain of apoMb (153 amino acid re sidues, no disulfide bonds) by several proteases (subtilisin, thermoly sin, chymotrypsin and trypsin) occurs at the level of chain segment 89 -96. In contrast, holoMb is resistant to proteolytic digestion when re acted under identical experimental conditions. Such selective proteoly sis implies that the F-helix of native holoMb (residues 82 to 97) is d isordered in apoMb, thus enabling binding and adaptation of this chain segment at the active site of the proteolytic enzymes for an efficien t peptide bond fission. That essentially only the F-helix in apoMb is largely disrupted was earlier inferred from spectroscopic measurements and molecular dynamics simulations. The results of this study provide direct experimental evidence for this and emphasize therefore that li mited proteolysis is a useful and reliable method for probing structur e and dynamics of proteins, complementing other experimental technique s such as NMR and X-ray crystallography. (C) 1997 Academic Press Limit ed.