Conformational heterogeneity of stability of apomyoglobin studied by hydrogen/deuterium exchange and electrospray ionization mass spectrometry.

The solution conformations and stability of apomyoglobin (apo-Mb), at both neutral and acidic pH, have been investigated by analyzing charge state distributions observed in the mass spectra, and by on-line monitoring of the hydrogen/deuterium (H/D) exchange using electrospray ionization mass spectrometry (ESI-MS) in combination with circular dichroism (CD). The results demonstrate that the conformation of apo-Mb, which lacks the heme group, is considerably less stable than that of holomyoglobin in identical solution conditions at neutral pH. ESI-MS shows that apo-Mb in the buffered solution at pH 7 (native state) and at pH 4.3 (intermediate state) yields two distinct charge state distributions that presumably correspond to different protein conformations. Both conformations have the same H/D exchange rate. This provides strong evidence that both the native and the intermediate state of apo-Mb have highly dynamic structures, consisting of two or more rapidly interconverting conformations rather than single fixed conformations. However, the H/D exchange rate of the acid-induced compact state of apo-Mb at pH approximately 2 [Goto, Y., Calciano, L. J., & Fink, A. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 573-577] indicates that it has a stable, partially folded conformation. Although CD data suggest that apo-Mb in H20 at pH 6 and in the buffered solution at pH 7 has a native-like secondary structure, the charge state distribution and the H/D exchange rate measurements indicate that a large portion of the apo-Mb molecules are unfolded or partially unfolded under these conditions. Thus, conformational information obtained from ESI-MS measurements of the charge state distributions and H/D exchange rates is complementary to that obtained from CD measurements. The combination of these three measurements can be used to assess the conformational stability and conformational heterogeneity of a protein.