Institute of Microbiology, v.v.i., Czech Academy of Sciences , 14220 Prague, Czech Republic.
Department of Biochemistry, Faculty of Science, Charles University in Prague , 12843 Prague, Czech Republic.
Anal Chem. 2018 Jan 16;90(2):1104-1113. doi: 10.1021/acs.analchem.7b02863. Epub 2018 Jan 2.
Chemical cross-linking coupled with mass spectrometry is a popular technique for deriving structural information on proteins and protein complexes. Also, cross-linking has become a powerful tool for stabilizing macromolecular complexes for single-particle cryo-electron microscopy. However, an effect of cross-linking on protein structure and function should not be forgotten, and surprisingly, it has not been investigated in detail so far. Here, we used kinetic studies, mass spectrometry, and NMR spectroscopy to systematically investigate an impact of cross-linking on structure and function of human carbonic anhydrase and alcohol dehydrogenase 1 from Saccharomyces cerevisiae. We found that cross-linking induces rather local structural disturbances and the overall fold is preserved even at a higher cross-linker concentration. The results establish general experimental conditions for chemical cross-linking with minimal effect on protein structure and function.
化学交联结合质谱是一种用于获取蛋白质和蛋白质复合物结构信息的常用技术。此外，交联已成为稳定用于单颗粒冷冻电子显微镜的大分子复合物的有力工具。然而，不应忘记交联对蛋白质结构和功能的影响，令人惊讶的是，到目前为止，还没有对此进行详细研究。在这里，我们使用动力学研究、质谱和 NMR 光谱法系统地研究了交联对人碳酸酐酶和酿酒酵母醇脱氢酶 1 的结构和功能的影响。我们发现交联会引起相对局部的结构干扰，即使在更高的交联剂浓度下，整体折叠也得以保持。这些结果为化学交联建立了一般的实验条件，对蛋白质结构和功能的影响最小。
