The 28-111 disulfide bond constrains the #alpha#-lactalbumin molten globule and weakens its cooperativity of folding

摘要

Our aim is to determine whether the disul- fide bonds of #alpha#-lactalbumin account for the lack of coopera- tive folding behavior reported for some molten globule vari- ants, in contrast to the highly cooperative folding reported for the PH 4 molten globule of apomyoglobin. Two different #alphta#lactalbumin genetic constructs are studied: [28-111], which has a single disulfide bond connecting two segments of the #alpha#-helix domain, and [all-Ala], which has no disulfide bonds. The superposition test used earlier to probe for coop- eretive folding of the apomyoglobin molten globule is used to determine whether there is an important difference in folding cooperativity between the molten globules of [28-lll] and [all-Ala] . The [all-Ala] construct behaves in the same manner as the apomyoglobin molten globule: its folding satisfies the superposition test in the three sets of anion conditions studied, and anions stabilize it against urea unfolding. The [28-111] construct behaves differently in both respects: the folding of its molten globule does not satisfy the superposition test in two ofthe three sets of anion conditions, and anions barely affect its stability. The 28-lll disulfide bond stabilizes the molten globule substantially, as expected from earlier work. Com- parison of the unfolding transition curves monitored by circular dichroism also demonstrates that [28-111] folds in a less cooperative manner than [all-Ala] : the unfolding curve of [28-lll] is significantly broader. Moreover, the unfolding curves indicate that [28-lll] has a lower helix content than [all-Ala]