Using nuclear magnetic resonance spectroscopy to study molten globule states of proteins.
Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for the study of the structure, dynamics, and folding of proteins in solution. It is particularly powerful when applied to dynamic or flexible systems, such as partially folded molten globule states of proteins, which are not usually amenable to X-ray crystallography. In this article, NMR methods suitable for the detailed characterisation of molten globule states are described. The specific method used to study the molten globule is determined by the quality of the NMR spectrum obtained. Molten globules are characterised by significant levels of secondary structure. Site-specific hydrogen-deuterium exchange experiments can be used to identify residues located in regions of secondary structure in the molten globule. If spectra characterised by sharp peaks are observed for the molten globule then information about secondary structure can be obtained by analysis of (1)H(alpha), (13)C(alpha), (13)C(beta), and (13)CO chemical shifts; this can be supplemented by (15)N relaxation studies. For molten globules characterised by extremely broad peaks (15)N-edited NMR experiments carried out in increasing concentrations of denaturants can be used to study the relative stabilities of different regions of structure. Examples of the application of these methods to the study of the low pH molten globule states of alpha-lactalbumin and apomyoglobin are presented.