A partly folded form (I) of apomyoglobin has an a-helix content of about 35%; in an earlier study, hydrogen exchange revealed that the A, G, and H helices are folded, while much of the rest of the protein is not [Hughson, F. M., Wright, P. E., & Baldwin, R. L. (1990) Science 249, 1544–1548]. Because A, G, and H form a compact subdomain in native myoglobin, we proposed that nativelike packing interactions among the three helices might be retained in the I form of apomyoglobin. To test this proposal, disruptive mutations were introduced into the A-H and G-H helix packing sites. These mutations destabilize native apomyoglobin relative to I. In contrast, the stability of I is relatively insensitive to mutation; in particular, side-chain volume alone does not appear to be important. These results indicate that the I form is not stabilized by nativelike A•H and G•H packing interactions. In support of this we show that partly helical peptides derived from the G and H helix regions of myoglobin do not pair in solution. Since the isolated G and H peptides are at best only partly helical, some type of interaction must stabilize these helices in the I form. Small increases in the stability of I are seen when mutation introduces a side chain of increased nonpolar surface area. We suggest that I is stabilized by relatively nonspecific hydrophobic interactions that allow it to adapt easily to mutation. In this and other respects, I appears to conform to the “molten globule” model, with the caveat that only part of the polypeptide chain appears to participate in the globule.
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