Membrane association of a protein increases the rate, extent, and specificity of chemical cross-linking.
Membrane association of a protein increases the rate, extent, and specificity of chemical cross-linking.
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蛋白质的膜缔合增加了化学交联的速率、程度和特异性。
DOI：
10.1021/bi4007176
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发表时间：
2013
期刊：
影响因子：
2.9
通讯作者：
Weis,RobertM
中科院分区：
文献类型：
--
作者：
Mudiyanselage,AruniPKKKarunanayake;Yang,Meili;Accomando,LeeA-R;Thompson,LynmarieK;Weis,RobertM
许多细胞过程涉及膜相关蛋白之间的相互作用，这些相互作用通过膜结合得到加强。我们使用交联反应来比较蛋白质在溶液和膜表面相互作用的程度和特异性。天冬氨酸受体(一种参与细菌趋化作用的跨膜受体)的可溶细胞质片段的半胱氨酸突变体被用于与硫醇特定的氧化剂二胺形成二硫键，以及与三官能团马来酰亚胺TMEA的化学交联反应。Cf与膜的结合是通过其N-末端His标签与含有镍螯合脂质的小泡的结合而介导的，因此在有无小泡的情况下进行的交联反应的不同之处只在于Cf是与小泡结合还是在溶液中游离。对于整个CF中的多个半胱氨酸，膜缔合被证明增加了这些反应的速度和程度。交联性是指天然结构中相互靠近的半胱氨酸之间的交联性，也可以通过膜结合来增强。这些结果提供了膜结合增强蛋白质-蛋白质相互作用的实验证明，这是理解涉及膜相关蛋白质的过程的重要考虑因素。这些实验进一步证明了交联条件对这些反应的重要性，这些反应经常被用来探索蛋白质的结构和动力学，以及膜结合的潜力，以恢复膜相关蛋白质在交联研究中的天然相互作用。
Many cellular processes involve interactions between membrane-associated proteins, and those interactions are enhanced by membrane association. We have used cross-linking reactions to compare the extent and specificity of protein interactions in solution versus on a membrane surface. Cysteine mutants of a soluble cytoplasmic fragment (CF) of the aspartate receptor, a transmembrane receptor involved in bacterial chemotaxis, are used in disulfide bond formation with the thiol-specific oxidant diamide and chemical cross-linking reactions with the trifunctional maleimide TMEA. CF binding to membranes is mediated by its N-terminal His tag binding to vesicles containing a nickel-chelating lipid, so cross-linking reactions conducted in the presence and absence of vesicles differ only in whether CF is bound to the vesicles or is free in solution. For multiple Cys throughout the CF, membrane association is shown to increase the rate and extent of these reactions. Cross-linking specificity, which is measured as the preference for cross-linking between Cys near each other in the native structure, is also enhanced by membrane association. These results provide an experimental demonstration that membrane binding enhances protein–protein interactions, an important consideration for understanding processes involving membrane-associated proteins. The experiments further demonstrate the importance of cross-linking conditions for these reactions that are often used to probe protein structure and dynamics and the potential of membrane association to restore native interactions of membrane-associated proteins for cross-linking studies.