Conformational Heterogeneity and Stability of Apomyoglobin Studied by Hydrogen/Deuterium Exchange and Electrospray Ionization Mass Spectrometry

Abstract

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.

Equilibrium unfolding of proteins monitored by electrospray ionization mass spectrometry: distinguishing two-state from multi-state transitions

“…At pH 2.0 the maximum is slightly shifted back to 19 (data not shown). ESI mass spectra for aMb at around pH 7.0, 4.0 and 2.0 have been reported previously 31 and agree qualitatively with the spectra shown here. The slight differences from the spectra of that study are most likely to be due to differences in the instrumentation and solvent conditions.…”

“…A pronounced dependence of the ESI mass spectra on the salt concentration of the solvent has been reported for aMb. 31 The relative abundance for some representative aMb charge states as a function of pH is depicted in Fig. 8(a) and 8(b).…”

Kinetic Properties and Metal Content of the Metallo-β-lactamase CcrA Harboring Selective Amino Acid Substitutions

“…A mild set of ESI interface conditions was employed for the detection of noncovalent metal-protein complexes, i.e. low curtain gas (desolvation) pressure and a low orifice (declustering) potential (13)(14)(15). High purity nitrogen gas was used as the nebulizing gas.…”

Substrate binding and conformational changes of Clostridium glutamicum diaminopimelate dehydrogenase revealed by hydrogen/deuterium exchange and electrospray mass spectrometry

“…With the advent of electrospray ionization mass spectrometry (ESI-MS) has come the ability to investigate intact protein structures using mass spectrometry. The rates of hydrogen/deuterium (H/D) exchange have been monitored by ESI-MS to study denaturation of bovine ubiquitin and hen lysozyme (Katta & Chait, 1993), to characterize structural perturbations in proteins (Robinson et al, 1994), and to probe conformational heterogeneity and stability of apomyoglobin (Wang & Tang, 1996). These studies on intact proteins, which were carried out in solvents that are compatible with ESI-MS, do not provide high resolution information with regard to specific changes within portions of the protein.…”