Gopinath T, Nelson Sarah E D, Soller Kailey J, Veglia Gianluigi
Department of Chemistry and ‡Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota , Minneapolis, Minnesota 55455, United States.
J Phys Chem B. 2017 May 4;121(17):4456-4465. doi: 10.1021/acs.jpcb.7b03268. Epub 2017 Apr 20.
Proteins exist in ensembles of conformational states that interconvert on various motional time scales. High-energy states of proteins, often referred to as conformationally excited states, are sparsely populated and have been found to play an essential role in many biological functions. However, detecting these states is quite difficult for conventional structural techniques. Recent progress in solution NMR spectroscopy made it possible to detect conformationally excited states in soluble proteins and characterize them at high resolution. As for soluble proteins, integral or membrane-associated proteins populate different structural states often modulated by their lipid environment. Solid-state NMR spectroscopy is the method of choice to study membrane proteins, as it can detect both ground and excited states in their natural lipid environments. In this work, we apply newly developed H-detected N-HSQC type experiments under moderate magic angle spinning speeds to detect the conformationally excited states of phospholamban (PLN), a single-pass cardiac membrane protein that regulates Ca transport across sarcoplasmic reticulum membrane. In its unbound state, the cytoplasmic domain of PLN exists in equilibrium between a T state, which is membrane bound and helical, and an R state, which is membrane detached and unfolded. The R state is important for regulation of the sarcoplasmic reticulum Ca-ATPase, but also for binding to protein kinase A. By hybridizing H detected solution and solid-state NMR techniques, it is possible to detect and resolve the amide resonances of the R state of PLN in liquid crystalline lipid bilayers. These new methods can be used to study the conformationally excited states of membrane proteins in native-like lipid bilayers.
蛋白质以构象状态的集合形式存在，这些构象状态在不同的运动时间尺度上相互转换。蛋白质的高能状态，通常被称为构象激发态，其数量稀少，但已发现在许多生物学功能中起着至关重要的作用。然而，对于传统的结构技术来说，检测这些状态相当困难。溶液核磁共振波谱学的最新进展使得检测可溶性蛋白质中的构象激发态并对其进行高分辨率表征成为可能。至于可溶性蛋白质，整合蛋白或膜相关蛋白通常会形成不同的结构状态，这些状态常常受到其脂质环境的调节。固态核磁共振波谱学是研究膜蛋白的首选方法，因为它可以在天然脂质环境中检测基态和激发态。在这项工作中，我们应用新开发的在中等魔角旋转速度下的氢检测氮-异核单量子相干（N-HSQC）型实验，来检测磷酸受纳蛋白（PLN）的构象激发态，PLN是一种单次跨膜的心脏膜蛋白，可调节钙离子跨肌浆网膜的转运。在其未结合状态下，PLN的胞质结构域存在于一种T态和一种R态之间的平衡中，T态是膜结合且呈螺旋状的，R态是膜分离且未折叠的。R态对于肌浆网钙-ATP酶的调节很重要，而且对于与蛋白激酶A的结合也很重要。通过结合氢检测的溶液和固态核磁共振技术，可以在液晶脂质双层中检测并分辨出PLN的R态的酰胺共振。这些新方法可用于研究天然脂质双层中膜蛋白的构象激发态。
