Institute of Molecular Modeling and Simulation, University of Natural Resources and Life Sciences, Vienna, Austria.
CEITEC-MU, Masaryk University, Kamenice 753/5, Bohunice, Brno, Czech Republic.
Biochim Biophys Acta Proteins Proteom. 2018 Mar;1866(3):442-450. doi: 10.1016/j.bbapap.2017.11.012. Epub 2017 Dec 5.
Mutations of cysteine are often introduced to e.g. avoid formation of non-physiological inter-molecular disulfide bridges in in-vitro experiments, or to maintain specificity in labeling experiments. Alanine or serine is typically preferred, which usually do not alter the overall protein stability, when the original cysteine was surface exposed. However, selecting the optimal mutation for cysteines in the hydrophobic core of the protein is more challenging. In this work, the stability of selected Cys mutants of 14-3-3ζ was predicted by free-energy calculations and the obtained data were compared with experimentally determined stabilities. Both the computational predictions as well as the experimental validation point at a significant destabilization of mutants C94A and C94S. This destabilization could be attributed to the formation of hydrophobic cavities and a polar solvation of a hydrophilic side chain. A L12E, M78K double mutant was further studied in terms of its reduced dimerization propensity. In contrast to naïve expectations, this double mutant did not lead to the formation of strong salt bridges, which was rationalized in terms of a preferred solvation of the ionic species. Again, experiments agreed with the calculations by confirming the monomerization of the double mutants. Overall, the simulation data is in good agreement with experiments and offers additional insight into the stability and dimerization of this important family of regulatory proteins.
半胱氨酸的突变通常被引入，例如在体外实验中避免形成非生理的分子间二硫键，或者在标记实验中保持特异性。通常情况下，当原始半胱氨酸暴露在表面时，丙氨酸或丝氨酸是首选，因为它们通常不会改变整体蛋白质稳定性。然而，在蛋白质疏水核心中选择半胱氨酸的最佳突变更具挑战性。在这项工作中，通过自由能计算预测了 14-3-3ζ 的选定 Cys 突变体的稳定性，并将获得的数据与实验确定的稳定性进行了比较。计算预测和实验验证都表明突变体 C94A 和 C94S 显著失稳。这种失稳可以归因于形成疏水腔和亲水侧链的极性溶剂化。进一步研究了 L12E、M78K 双突变体的低二聚倾向。与天真的预期相反，这种双突变体并没有导致形成强盐桥，这可以根据离子物种的优先溶剂化来解释。同样，实验与计算结果一致，证实了双突变体的单体化。总体而言，模拟数据与实验结果非常吻合，并为这些重要的调节蛋白家族的稳定性和二聚化提供了额外的见解。
