Fiala Jan, Kukačka Zdeněk, Novák Petr
Institute of Microbiology of the Czech Academy of Sciences, Vestec, Czech Republic; Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
Institute of Microbiology of the Czech Academy of Sciences, Vestec, Czech Republic.
J Proteomics. 2020 Apr 30;218:103716. doi: 10.1016/j.jprot.2020.103716. Epub 2020 Feb 20.
The combination of chemical cross-linking and mass spectrometry is currently a progressive technology for deriving structural information of proteins and protein complexes. In addition, chemical cross-linking is a powerful tool for stabilizing macromolecular complexes for single particle cryo-electron microscopy. Broad pallets of cross-linking chemistry, currently available for the majority of cross-linking experiments, still rely on the amine-reactive N-hydroxysuccinimide esters targeting mainly N-termini and lysine side chains. These cross-linkers are divided into two groups: water soluble and water insoluble; and research teams prefer one or another speculating on the benefits of their choice. However, the effect of cross-linker polarity on the outcome of cross-linking reaction has never been studied. Herein, we use both polar (bis(sulfosuccinimidyl) glutarate) and non-polar (disuccinimidyl glutarate) cross-linkers and systematically investigated the impact of cross-linker hydrophobicity on resulting distance constraints, using bovine serum albumin as a model protein. SIGNIFICANCE: Even though the amine reactive BS2G and DSG cross-linkers have the same length of spacer and are based on N-hydroxysuccinimidic group, our data showed that each of them formed preferentially different cross-links. We demonstrated that the choice of cross-linker can have a significant impact on the output data for structural characterization of biomolecules. Using equimolar mixtures of DSG with d6-BS2G, and BS2G with d6-DSG, we established that the polar BS2G preferentially bound to polar regions of modified molecule, whereas non-polar DSG bound to hydrophobic regions. This phenomenon established that the mixture of polar and non-polar cross-linkers acted as an efficient tool for the determination of distance constraints in proteins.
化学交联与质谱联用目前是一种用于获取蛋白质和蛋白质复合物结构信息的先进技术。此外，化学交联是一种用于稳定单颗粒冷冻电子显微镜大分子复合物的强大工具。目前大多数交联实验可用的广泛交联化学试剂，仍然依赖于主要靶向N端和赖氨酸侧链的胺反应性N-羟基琥珀酰亚胺酯。这些交联剂分为两类：水溶性和水不溶性；研究团队根据其选择的益处而偏好其中一种。然而，交联剂极性对交联反应结果的影响从未被研究过。在此，我们使用极性（双（磺基琥珀酰亚胺基）戊二酸酯）和非极性（双琥珀酰亚胺基戊二酸酯）交联剂，并以牛血清白蛋白作为模型蛋白，系统地研究了交联剂疏水性对所得距离限制的影响。意义：尽管胺反应性BS2G和DSG交联剂具有相同长度的间隔臂且基于N-羟基琥珀酰亚胺基团，但我们的数据表明它们各自优先形成不同的交联。我们证明交联剂的选择对生物分子结构表征的输出数据可能有重大影响。使用DSG与d6-BS2G以及BS2G与d6-DSG的等摩尔混合物，我们确定极性BS2G优先结合到修饰分子的极性区域，而非极性DSG结合到疏水区域。这一现象表明极性和非极性交联剂的混合物是确定蛋白质中距离限制的有效工具。
