Elucidating in Vivo Structural Dynamics in Integral Membrane Protein by Hydroxyl Radical Footprinting
Elucidating in Vivo Structural Dynamics in Integral Membrane Protein by Hydroxyl Radical Footprinting
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DOI：
10.1074/mcp.m900081-mcp200
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发表时间：
2009-08-01
影响因子：
7
通讯作者：
Sze, Siu Kwan
中科院分区：
文献类型：
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作者：
Zhu, Yi;Guo, Tiannan;Sze, Siu Kwan
在这里，我们描述了一种新的足迹技术来探索膜蛋白的活体结构动力学。该方法利用原位产生的羟基自由基氧化和共价修饰活的大肠杆菌细胞表面的生物分子。膜蛋白组富集和纯化后，用串联质谱仪鉴定蛋白质的修饰氨基酸残基，以绘制蛋白质的溶剂可及表面，形成蛋白质体内结构的足迹。在鉴定的大约100个外膜蛋白中，我们研究了一个典型的β-桶状结构-Porin OmpF的结构细节。我们发现，在生理条件下，19个氨基酸的修饰可以重复性地检测到6个OmpF的修饰胰蛋白酶多肽。修饰后的氨基酸残基广泛分布在外环区、β链和周质转折区，根据结晶学数据证实它们都是溶剂可及的。我们进一步将这种方法扩展到利用模拟生理环境的变化来研究OmpF在体内的电压门控动力学，无论是pH还是离子强度。我们的数据首次显示了孔蛋白OmpF在体内的电压门控，并支持了孔小孔区域局部静电场变化可能改变孔蛋白通道切换的机制。因此，这种新的方法可以成为研究活细胞膜蛋白结构动力学的一种潜在的有效方法。分子与细胞蛋白质组学8：1999-2010,2009。
We describe here a novel footprinting technique to probe the in vivo structural dynamics of membrane protein. This method utilized in situ generation of hydroxyl radicals to oxidize and covalently modify biomolecules on living Escherichia coli cell surface. After enriching and purifying the membrane proteome, the modified amino acid residues of the protein were identified with tandem mass spectrometry to map the solvent-accessible surface of the protein that will form the footprint of in vivo structure of the protein. Of about 100 outer membrane proteins identified, we investigated the structure details of a typical beta-barrel structure, the porin OmpF. We found that six modified tryptic peptides of OmpF were reproducibly detected with 19 amino acids modified under the physiological condition. The modified amino acid residues were widely distributed in the external loop area, beta-strands, and periplasmic turning area, and all of them were validated as solvent-accessible according to the crystallography data. We further extended this method to study the dynamics of the voltage gating of OmpF in vivo using mimic changes of physiological circumstance either by pH or by ionic strength. Our data showed the voltage gating of porin OmpF in vivo for the first time and supported the proposed mechanism that the local electrostatic field changes in the eyelet region may alter the porin channels to switch. Thus, this novel method can be a potentially efficient method to study the structural dynamics of the membrane proteins of a living cell. Molecular & Cellular Proteomics 8: 1999-2010, 2009.