Cl. Chyan et al., STRUCTURE AND STABILITY OF THE MOLTEN GLOBULE STATE OF GUINEA-PIG ALPHA-LACTALBUMIN - A HYDROGEN-EXCHANGE STUDY, Biochemistry, 32(21), 1993, pp. 5681-5691
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.