STRUCTURE AND STABILITY OF THE MOLTEN GLOBULE STATE OF GUINEA-PIG ALPHA-LACTALBUMIN - A HYDROGEN-EXCHANGE STUDY

A partially folded state of guinea pig alpha-lactalbumin (the A-state or molten globule state), formed by denaturation at low pH, has been s tudied using hydrogen exchange methods. The overall distribution of ex change kinetics, measured by 1-D NMR, suggests that fewer than 20 amid es in the structure are involved in highly persistent residual structu re, although CD results suggest that many other parts of the chain are folded, for a significant proportion of the time, into less stable st ructural 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, o f those that are slow to exchange in the A-state retain that protectio n in the native state. This suggests that the persistent structure in the A-state is native-like although the possibility that nonnative lik e structural elements persist cannot be eliminated. Resonance assignme nts for key residues in the NMR spectrum of the native state have enab led 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 hydropho bic segments, corresponding to the B- and C-helices of the native stru cture. This strongly suggests that the most persistent structure of th e A-state includes these regions. A variety of lines of evidence, incl uding 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 th e native structure persists in the A-state, although without the stere ochemical rigidity of the native tertiary structure. The other domain of the native structure, including the beta-sheet, appears not to cont ain structural elements which persist to the same extent in the A-stat e, emphasizing that the molten globule is highly heterogeneous, in ter ms of the stability and specificity of both backbone and side chain in teractions.