A partially folded state of guinea pig alpha-lactalbumin (the A-state or molten globule state), formed by denaturation at low pH, has been studied using hydrogen exchange methods. The overall distribution of exchange kinetics, measured by 1-D NMR, suggests that fewer than 20 amides in the structure are involved in highly persistent residual structure, although CD results suggest that many other parts of the chain are folded, for a significant proportion of the time, into less stable structural elements. The pH-jump experiments show that some amides that are strongly protected from exchange in the native state become freely accessible in the A-state but that conversely a majority, at least, of those that are slow to exchange in the A-state retain that protection in the native state. This suggests that the persistent structure in the A-state is native-like although the possibility that nonnative like structural elements persist cannot be eliminated. Resonance assignments for key residues in the NMR spectrum of the native state have enabled us to use the pH-jump method also to identify the majority of the most protected amides in the A-state: they are located in two hydrophobic segments, corresponding to the B- and C-helices of the native structure. This strongly suggests that the most persistent structure of the A-state includes these regions. A variety of lines of evidence, including fluorescence quenching data and, most remarkably, very effective protection from exchange of an indole NH in a tryptophan side chain, suggest that some form of hydrophobic core in the helical domain of the native structure persists in the A-state, although without the stereochemical rigidity of the native tertiary structure. The other domain of the native structure, including the beta-sheet, appears not to contain structural elements which persist to the same extent in the A-state, emphasizing that the molten globule is highly heterogeneous, in terms of the stability and specificity of both backbone and side chain interactions.

'H-NMR assignments have been defined for the aromatic-ring protons of the bovine, guinea pig and human variants of a-lactalbumin. Spin-system networks were identified by means of doublequantum-filtered two-dimensional J-correlated spectroscopy and two-dimensional relayed coherence spectroscopy data. Analysis of two-dimensional nuclear-Overhauser-enhancement spectroscopy data of the proteins indicated that in each case two clusters of aromatic residues exist. The two clusters are also evident in the crystal structure of the human protein, and this evidence, in conjunction with sequence differences between the three proteins, permitted sequence-specific assignments to be made for the majority of aromatic residues. Remaining ambiguities in the assignments could be resolved by analysis of photochemically induced dynamic nuclear polarization (PCIDNP) effects. Comparison of the PCIDNP spectra of the three proteins indicated the presence of only minor differences in the surface exposure of conserved aromatic residues. Taken together, these results indicate that the environments of the conserved aromatic residues in bovine, guinea pig and human a-lactalbumin in solution are very similar to each other, and that the solution and the crystal forms of at least the human protein are similar.