Pulsed hydrogen/deuterium exchange mass spectrometry for time-resolved membrane protein folding studies
Pulsed hydrogen/deuterium exchange mass spectrometry for time-resolved membrane protein folding studies
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DOI：
10.1002/jms.3127
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发表时间：
2012-12-01
影响因子：
2.3
通讯作者：
Konermann, Lars
中科院分区：
文献类型：
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作者：
Khanal, Anil;Pan, Yan;Konermann, Lars
通过脉冲氢/氘交换（HDX）质谱（MS）进行的动力学折叠实验是用于水溶性蛋白质的成熟工具。据我们所知，目前的研究是第一个将这种方法应用于整合膜蛋白的研究。细菌视紫红质（BR）的天然状态包括七个跨膜螺旋和共价结合的视网膜辅因子。BR暴露于十二烷基硫酸钠（SDS）诱导部分展开和视网膜丢失。我们采用定制的三级混合装置进行BR重折叠的脉冲HDX/MS调查。通过将SDS变性蛋白质与双胞混合来触发反应。经过可变的折叠时间（10？M到24？h）在快速交换条件下，将蛋白质暴露于过量的D2 O缓冲液。HDX脉冲终止酸淬火后24？随后通过尺寸排阻色谱法和电喷雾MS进行离线分析。这些测量产生作为折叠时间的函数的受保护骨架NH位点的数目，反映二级结构的恢复。我们的研究结果表明，BR的二级结构形成相当晚的反应过程中，在10？S和超越。人们希望，在未来将有可能扩展脉冲HDX/MS方法在这里采用的膜蛋白比BR。版权所有（c）2012约翰威利父子有限公司
Kinetic folding experiments by pulsed hydrogen/deuterium exchange (HDX) mass spectrometry (MS) are a well-established tool for water-soluble proteins. To the best of our knowledge, the current study is the first that applies this approach to an integral membrane protein. The native state of bacteriorhodopsin (BR) comprises seven transmembrane helices and a covalently bound retinal cofactor. BR exposure to sodium dodecyl sulfate (SDS) induces partial unfolding and retinal loss. We employ a custom-built three-stage mixing device for pulsed-HDX/MS investigations of BR refolding. The reaction is triggered by mixing SDS-denatured protein with bicelles. After a variable folding time (10?ms to 24?h), the protein is exposed to excess D2O buffer under rapid exchange conditions. The HDX pulse is terminated by acid quenching after 24?ms. Subsequent off-line analysis is performed by size exclusion chromatography and electrospray MS. These measurements yield the number of protected backbone NH sites as a function of folding time, reflecting the recovery of secondary structure. Our results indicate that much of the BR secondary structure is formed quite late during the reaction, on a time scale of 10?s and beyond. It is hoped that in the future it will be possible to extend the pulsed-HDX/MS approach employed here to membrane proteins other than BR. Copyright (c) 2012 John Wiley & Sons, Ltd.