Initial disulfide formation steps in the folding of an omega-conotoxin.
Initial disulfide formation steps in the folding of an omega-conotoxin.
复制标题
omega-芋螺毒素折叠过程中的初始二硫键形成步骤。
作者：
M. Price;G. Bulaj;D. Goldenberg
为了确定omega-conooxin的天然二硫化物是否在这些小蛋白的折叠早期优先稳定，我们测量了omega-conooxoxin MVIIA的三个类似物的二硫键形成速率和平衡。在每个类似物中，三对二硫键半胱氨酸残基中的一对被丙氨酸残基取代，剩下四个半胱氨酸残基，可以与一个二硫键形成六个中间体，三个物种与两个二硫键形成中间体。对于每个类似物，鉴定了所有二硫键物种，并测量了通过与氧化和还原的谷胱甘肽交换形成单个物种的平衡常数。这些平衡常数代表半胱氨酸硫醇的有效浓度，在完全还原的蛋白质中的范围从0.01到0.4M。加入8M尿素后，几乎没有形成天然二硫化物的倾向，形成第一和第二二硫化物的平衡仅略有下降。四个半胱氨酸类似物的数据，以及六个半胱氨酸形式的平衡数据，也被用来估计一旦存在两个天然二硫化物就形成第三个二硫化物的有效浓度。在0和8M尿素存在下，这些有效浓度分别约为100M和10M。结果表明，在两个二硫键形成之前，这些分子在折叠过程中很少或没有优先形成天然相互作用，此后天然相互作用之间具有高度的协同性。
To determine whether the native disulfides of omega-conotoxins are preferentially stabilized early in the folding of these small proteins, the rates and equilibria for disulfide formation were measured for three analogues of omega-conotoxin MVIIA. In each analogue, one of the three pairs of disulfide-bonded Cys residues was replaced with Ala residues, leaving four Cys residues that can form six intermediates with one disulfide and three species with two disulfides. For each analogue, all of the disulfide-bonded species were identified, and the equilibrium constants for forming the individual species via exchange with oxidized and reduced glutathione were measured. These equilibrium constants represent effective concentrations of the Cys thiols and ranged from 0.01 to 0.4 M in the fully reduced protein. There was little or no preference for forming the native disulfides, and the equilibria for forming the first and second disulfides decreased only slightly upon the addition of 8 M urea. The data for the four-Cys analogues, together with equilibrium data for the six-Cys form, were also used to estimate effective concentrations for forming a third disulfide once two native disulfides are present. These effective concentrations were approximately 100 and 10 M in the presence of 0 and 8 M urea, respectively. The results indicate that there is little or no preferential formation of native interactions in the folding of these molecules until two disulfides have formed, after which there is a high degree of cooperativity among the native interactions.