Price-Carter M, Hull M S, Goldenberg D P
Department of Biology, University of Utah, Salt Lake City 84112-0840, USA.
Biochemistry. 1998 Jul 7;37(27):9851-61. doi: 10.1021/bi9803978.
Although it contains only 25 amino acid residues, omega-conotoxin MVIIA folds into a well-defined three-dimensional structure that is stabilized by 3 disulfide bonds. To assess the contributions of the disulfides to folding and stability, three analogues, each with one pair of disulfide-bonded Cys residues replaced with Ala, were prepared and characterized. The analogues also contained a C-terminal Gly residue that is believed to be present when the peptide folds in vivo and has been shown previously to stabilize the native structure. Circular dichroism spectra and biological assays of the analogues indicated that removing any one of the disulfides greatly destabilized the native conformation. The two disulfides in each analogue were also reduced much more rapidly than in the native form with three disulfides. When the analogues were fully reduced and allowed to form disulfides in the presence of oxidized and reduced glutathione, the native disulfides were not formed in preference to non-native disulfides, further indicating that the forms with two-native disulfides are not significantly stabilized by noncovalent interactions. However, the measured equilibrium constants for disulfide formation indicate that forming any two of the three native disulfides leads to an effective concentration of approximately 25-50 M for the two remaining thiols. The two-disulfide analogues thus appear to represent a stage of folding in which the polypeptide is constrained to a distribution of relatively compact conformations that greatly favor formation of the third disulfide and the final folded structure.
尽管ω-芋螺毒素MVIIA仅含有25个氨基酸残基，但它折叠成一个由3个二硫键稳定的明确三维结构。为了评估二硫键对折叠和稳定性的贡献，制备并表征了三种类似物，每种类似物都有一对二硫键连接的半胱氨酸残基被丙氨酸取代。这些类似物还含有一个C端甘氨酸残基，据信该残基在肽在体内折叠时存在，并且先前已证明其能稳定天然结构。类似物的圆二色光谱和生物学分析表明，去除任何一个二硫键都会极大地破坏天然构象的稳定性。与具有三个二硫键的天然形式相比，每种类似物中的两个二硫键也被更快地还原。当类似物完全还原并在氧化型和还原型谷胱甘肽存在下形成二硫键时，天然二硫键并非优先于非天然二硫键形成，这进一步表明具有两个天然二硫键的形式并没有通过非共价相互作用得到显著稳定。然而，测得的二硫键形成平衡常数表明，形成三个天然二硫键中的任意两个会导致其余两个硫醇的有效浓度约为25 - 50 M。因此，两个二硫键的类似物似乎代表了折叠的一个阶段，在此阶段多肽被限制在相对紧凑构象的分布中，这极大地有利于第三个二硫键的形成和最终的折叠结构。
