Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry.
Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry.
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DOI：
10.1021/acs.analchem.7b01310
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发表时间：
2017-09-05
影响因子：
7.4
通讯作者：
Henderson PJF
中科院分区：
文献类型：
--
作者：
Calabrese AN;Jackson SM;Jones LN;Beckstein O;Heinkel F;Gsponer J;Sharples D;Sans M;Kokkinidou M;Pearson AR;Radford SE;Ashcroft AE;Henderson PJF
Cys 可及性和定量完整质谱 (MS) 分析旨在研究 Mhp1 的拓扑转变，Mhp1 是液化微杆菌乙内酰脲钠连接转运的膜蛋白。 Mhp1 已结晶为三种形式（面向外的开放型、面向外的与底物结合的闭塞型和向内的开放型）。我们发现，三个天然半胱氨酸残基之一 Cys327 在向内（相对于向外）形式中具有增强的溶剂可及性。纯化的蛋白质在去污剂中与硫醇反应性 N-乙基马来酰亚胺 (NEM) 发生反应，导致 Cys327 发生修饰，表明 Mhp1 主要采用向内构象。添加钠离子或底物 5-苄基-L-乙内酰脲 (L-BH) 不会改变这种构象平衡，但系统地同时添加两者会导致标记减弱，表明向外向构象的转变，这可以使用传统的酶动力学分析来解释。这种测量可以提供每个配体的 Km 以及离子-底物耦合构象变化的化学计量。扰乱底物结合位点的突变要么导致蛋白质无法采用面向外的构象，要么导致结构整体不稳定。该方法将共价标记、质谱和动力学分析结合在适用于一系列系统的简单工作流程中，从而能够询问在不同浓度的底物下发挥功能所需的蛋白质构象的变化，以及突变对这些构象转变的影响。
Cys accessibility and quantitative intact mass spectrometry (MS) analyses have been devised to study the topological transitions of Mhp1, the membrane protein for sodium-linked transport of hydantoins from Microbacterium liquefaciens. Mhp1 has been crystallized in three forms (outward-facing open, outward-facing occluded with substrate bound, and inward-facing open). We show that one natural cysteine residue, Cys327, out of three, has an enhanced solvent accessibility in the inward-facing (relative to the outward-facing) form. Reaction of the purified protein, in detergent, with the thiol-reactive N-ethylmalemide (NEM), results in modification of Cys327, suggesting that Mhp1 adopts predominantly inward-facing conformations. Addition of either sodium ions or the substrate 5-benzyl-l-hydantoin (L-BH) does not shift this conformational equilibrium, but systematic co-addition of the two results in an attenuation of labeling, indicating a shift toward outward-facing conformations that can be interpreted using conventional enzyme kinetic analyses. Such measurements can afford the Km for each ligand as well as the stoichiometry of ion–substrate-coupled conformational changes. Mutations that perturb the substrate binding site either result in the protein being unable to adopt outward-facing conformations or in a global destabilization of structure. The methodology combines covalent labeling, mass spectrometry, and kinetic analyses in a straightforward workflow applicable to a range of systems, enabling the interrogation of changes in a protein’s conformation required for function at varied concentrations of substrates, and the consequences of mutations on these conformational transitions.
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DOI：
10.1016/j.ymeth.2015.02.018
发表时间：
2015-11-01
期刊：
Methods (San Diego, Calif.)
影响因子：
--
作者：
Calabrese AN;Ault JR;Radford SE;Ashcroft AE
通讯作者：
Ashcroft AE
影响因子：
14.8
作者：
Guan, Lan;Kaback, H. Ronald
通讯作者：
Kaback, H. Ronald
影响因子：
7.4
作者：
Calabrese AN;Watkinson TG;Henderson PJ;Radford SE;Ashcroft AE
通讯作者：
Ashcroft AE
影响因子：
16.8
作者：
Kazmier K;Sharma S;Quick M;Islam SM;Roux B;Weinstein H;Javitch JA;McHaourab HS
通讯作者：
McHaourab HS
影响因子：
3.4
作者：
Adelman, Joshua L.;Dale, Amy L.;Grabe, Michael
通讯作者：
Grabe, Michael