Department of Pharmaceutical Chemistry, Multidisciplinary Research Building, The University of Kansas, 2030 Becker Drive, Lawrence, Kansas 66047, USA.
J Pharm Sci. 2010 Jun;99(6):2643-54. doi: 10.1002/jps.22055.
It is well established that the oxidation state of cysteine residues in proteins is critical to overall physical stability. Disulfide bonds most often impart thermodynamic stability, but in some cases, diminish it. Predicting the circumstances that lead to each outcome is difficult because mechanistic information is lacking. Because the techniques typically used to study protein stability do not provide sufficient detail, high-resolution NMR was used in combination with low-resolution analysis to obtain mechanistic information regarding disulfide bond formation in a model protein. T(m) (CD) and T(onset) (SLS) for the reduced and oxidized wild type and C104S and C49S mutants were measured. The mutant proteins have altered T(m)s and T(onset)s compared to the reduced wild type, indicating that differences in local interactions of the Cys side chains are important for stability. The NMR spectra clearly show distinct differences in the chemical environment surrounding these Cys residues and the overall tertiary structure. The C49S protein, which is less stable and more aggregation prone than reduced wild type, lacks a hydrogen bond between Y53 and H103. Increased flexibility of the Y53-containing loop is correlated with increased dynamics and unraveling of alpha2, which likely leads to edge strand initiated aggregation of the central beta-sheet.
众所周知，蛋白质中半胱氨酸残基的氧化态对整体物理稳定性至关重要。二硫键通常赋予热力学稳定性，但在某些情况下会降低其稳定性。由于缺乏机制信息，预测导致每种结果的情况很困难。由于用于研究蛋白质稳定性的技术通常不能提供足够的细节，因此使用高分辨率 NMR 结合低分辨率分析来获得有关模型蛋白质中二硫键形成的机制信息。测量了还原型和氧化型野生型以及 C104S 和 C49S 突变体的 T(m)(CD)和 T(onset)(SLS)。与还原型野生型相比，突变蛋白的 T(m)和 T(onset)发生了变化，这表明 Cys 侧链的局部相互作用的差异对稳定性很重要。NMR 谱清楚地显示了这些 Cys 残基周围的化学环境和整体三级结构的明显差异。C49S 蛋白比还原型野生型更不稳定且更容易聚集，缺乏 Y53 和 H103 之间的氢键。含有 Y53 的环的柔韧性增加与α2 的解旋和展开相关，这可能导致边缘链开始聚集中心β-折叠。
