The human alpha-lactalbumin molten globule: comparison of structural preferences at pH 2 and pH 7

Structural investigations of molten globules provide an important contribution towards understanding protein folding pathways. A close similarity between equilibrium molten globule states and kinetic species observed during refolding has been reported for several proteins. However, the experimental conditions, and in particular the pH, under which the equilibrium and kinetic species are studied often differ significantly. For human alpha-lactalbumin (alpha-LA), the equilibrium molten globule is most often studied at pH 2, the so-called A-state, while kinetic refolding experiments are performed at neutral pH. alpha-LA contains a large number of acidic amino acid residues that may influence the properties of the molten globule differently at low and neutral pH. In this study, we investigate the structural preferences of the alpha-LA molten globule at pH 7 at the level of individual residues using nuclear magnetic resonance spectroscopy and compare these data with previous results obtained at pH 2. We show that differences exist in the conformational ensemble that describes the alpha-LA molten globule at these two pH values. The molten globule at pH 7 is generally less stable than that at the low pH A-state. Most notable are differences in the stability of structure for the C-helix and the calcium-binding loop that precedes it and differences in the contribution of long-range hydrophobic contacts between the N-terminal and C-terminal regions of the alpha-domain to the stability of the molten globule. Our results are discussed in the context of previous studies of the alpha-LA molten globule and can be used to reconcile apparent discrepancies in published data relating to the C-helix. In the light of our results, the low pH A-state may not be the best model for the kinetic molten globule observed during refolding of alpha-LA. The pH-dependent effects reported here for alpha-LA may be of relevance in comparisons of equilibrium and kinetic molten globules of other proteins.