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首页> 外文期刊>Biochemistry >Hydrogen Exchange Mass Spectrometry of Functional Membrane-Bound Chemotaxis Receptor Complexes
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Hydrogen Exchange Mass Spectrometry of Functional Membrane-Bound Chemotaxis Receptor Complexes

机译：功能膜结合趋化性络合物的氢交换质谱

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The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (～2 ?) piston displacement of one helix of the periplasmic and transmembrane domains toward the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 ? away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen?deuterium exchange mass spectrometry (HDX-MS) measurements of global exchange of the CF demonstrate that the CF exhibits significantly slower exchange in functional complexes than in solution. Because the exchange rates in functional complexes are comparable to those of other proteins with similar structures, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system.
机译：认为细菌趋化性受体的跨膜信号传导机制涉及受体构象和动力学的变化。受体用两种其他蛋白质，凿孔和咀嚼的三元复合物起作用，其形成延伸的膜结合阵列。以前的研究表明，引诱剂结合诱导一个螺旋状和跨膜结构域的一个螺旋的小（〜2？）活塞位移朝向细胞质，但目前尚不清楚该信号如何通过细胞质结构域传播以控制激酶活性Chea在膜 - 远端尖端束缚，近200？离开。细胞质结构域已被证明是高度动态的，这提出了如何通过动态结构域传播的小活塞运动如何控制Chea激酶活性。为了解决这一点，我们开发了一种用Chea和Chew在功能络合物中测量受体细胞质片段（CF）的动态的方法。氢气？氘交换质谱（HDX-MS）CF的全局交换的测量表明，CF在功能复合物中表现出显着较慢的交换。因为功能复合物中的汇率与具有相似结构类似的其他蛋白质的汇率相当，所以CF似乎是这些复合物中的结构良好的蛋白质，其与其在传播似乎是微小构象变化的信号中的作用兼容在受体的周质和跨膜结构域中。我们还通过掺入胃蛋白酶消化步骤以产生具有87％序列覆盖率的肽和仅20％的返回交换来证明本次方案对本地交换测量的可行性。该方法将HDX-MS延伸到膜结合的函数复合物，没有可扰乱系统的稳定性或结构的洗涤剂。

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