Is apomyoglobin a molten globule? Structural characterization by NMR

Multi-dimensional heteronuclear NMR spectroscopy has been used to obtain structural information on isotopically labeled recombinant sperm whale apomyoglobin in the native state at pH 6.1. Assignments for backbone resonances ((HN)-H-1, N-15, and C-13(alpha)) have been made for a large fraction of the residues in the protein. The secondary structure indicated by the observed chemical shifts is nearly identical to that found in carbonmonoxy-holomyoglobin in all assigned regions. In addition the chemical shifts themselves are highly similar in both proteins. This suggests that the majority of the apomyoglobin polypeptide chain adopts a well defined structure which is very similar to that of holomyoglobin. However, backbone resonances from a contiguous region of the apoprotein, corresponding to the EF loop, the F helix, the FG loop, and the beginning of the G helix, are broadened beyond detection due to conformational fluctuations. We propose that the polypeptide in this region exchanges between a holoprotein-like conformation and one or more unfolded or partially folded states. Such a model can explain the current NMR data, the charge state distributions observed by mass spectrometry, and the effects of mutagenesis. Apomyoglobin possesses many of the characteristics of a native, globular protein and does not adhere to the classical description of a molten globule.