Quezada Cindy M, Schulman Brenda A, Froggatt Joanna J, Dobson Christopher M, Redfield Christina
Chemistry Research Laboratory, Oxford Centre for Molecular Sciences, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK.
J Mol Biol. 2004 Apr 16;338(1):149-58. doi: 10.1016/j.jmb.2004.02.045.
NMR spectroscopy has been used to follow the urea-induced unfolding of the low pH molten globule states of a single-disulfide variant of human alpha-lactalbumin ([28-111] alpha-LA) and of two mutants, each with a single proline substitution in a helix. [28-111] alpha-LA forms a molten globule very similar to that formed by the wild-type four-disulfide protein, and this variant has been used as a model for the alpha-lactalbumin (alpha-LA) molten globule in a number of studies. The urea-induced unfolding behavior of [28-111] alpha-LA is similar to that of the four-disulfide form of the protein, except that [28-111] alpha-LA is less stable and has greater cooperativity in the loss of different elements of structure. For one mutant, L11P, the helix containing the mutation is highly destabilized such that it is completely unfolded even in the absence of urea. By contrast, for the other mutant, Q117P, the helix containing the mutation retains its compact structure. Both mutations, however, show significant long-range destabilization of the overall fold showing that the molten globule state has a degree of global cooperativity. The results reveal that different permutations of three of the four major alpha-helices of the protein can form a stable, locally cooperative, compact structural core. Taken together, these findings demonstrate that the molten globule state of alpha-LA is an ensemble of conformations, with different subsets of structures linked by a range of long-range interactions.
核磁共振光谱已被用于追踪尿素诱导的人α-乳白蛋白单二硫键变体（[28-111]α-LA）以及两个突变体（每个突变体在一个螺旋中有一个脯氨酸取代）的低pH熔融球状态的去折叠过程。[28-111]α-LA形成的熔融球状态与野生型四二硫键蛋白形成的非常相似，并且在许多研究中该变体已被用作α-乳白蛋白（α-LA）熔融球状态的模型。[28-111]α-LA的尿素诱导去折叠行为与该蛋白的四二硫键形式相似，只是[28-111]α-LA稳定性较差，并且在结构不同元件的丧失方面具有更大的协同性。对于一个突变体L11P，包含该突变的螺旋高度不稳定，以至于即使在没有尿素的情况下它也完全去折叠。相比之下，对于另一个突变体Q117P，包含该突变的螺旋保留其紧密结构。然而，这两个突变都显示出整体折叠的显著远程不稳定，表明熔融球状态具有一定程度的全局协同性。结果表明，该蛋白四个主要α-螺旋中的三个的不同排列可以形成一个稳定的、局部协同的、紧密的结构核心。综上所述，这些发现表明α-LA的熔融球状态是一组构象，不同的结构子集通过一系列远程相互作用相连。
