alpha-lactalbumin forms a compact molten globule in the absence of disulfide bonds

Human alpha-lactalbumin (alpha-LA) is a four disulfide-bonded protein that adopts partially structured conformations under a variety of mildly denatur ing conditions. At low pH, the protein is denatured but compact, with a hig h degree of secondary structure and a native-like fold. This is commonly re ferred to as a molten globule, A variant of alpha-LA, in which all eight cy steines have been mutated to alanine (all-Ala alpha-LA), has been studied u sing NMR spectroscopy. At low pH all-Ala alpha-LA is nearly as compact as w ild type alpha-LA. Urea-induced unfolding experiments reveal that the resid ues that remain compact in the absence of disulfide bonds are those that ar e most resistant to unfolding in the wild-type alpha-LA molten globule. Thi s is particularly remarkable because this stable core is formed by segments of the polypeptide chain from both the N- and C-termini, These results sho w that the overall architecture of the protein fold of alpha-LA is determin ed by the polypeptide sequence itself, and not as the result of crosslinkin g by disulfide bonds, and provide insight into the way in which the sequenc e codes for the fold.