Arora Ashish
Molecular and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, UP, India.
Methods Mol Biol. 2013;974:389-413. doi: 10.1007/978-1-62703-275-9_17.
Several recent advancements have transformed solution NMR spectroscopy into a competitive, elegant, and eminently viable technique for determining the solution structures of membrane proteins at the level of atomic resolution. Once a good level of cell-based or cell-free expression and purification of a suitably sized membrane protein has been achieved, then NMR offers a combination of several versatile strategies, for example, choice of appropriate deuterated or non-deuterated detergents, temperature, and ionic strength; isotope labelling with (2)H, (13)C, (15)N, with or without protonation of Ile (δ1), Leu, and Val methyl protons; combinatorial labelling of specific amino acids; transverse relaxation-optimized NMR spectroscopy-based, Nonuniform sampling-based, and other NMR experiments; measurement of residual dipolar couplings using stretched polyacrylamide gels or DNA nanotubes; and spin-labelling and paramagnetic relaxation enhancements. Strategic combinations of these advancements together with availability of highly sensitive cryogenically cooled probes equipped high-field NMR spectrometers (up to 1 GHz (1)H frequency) have allowed the perseverant investigator to successfully overcome several of the conventional pitfalls associated with the NMR technique and membrane proteins, viz., low sensitivity, poor sample stability, spectral crowding, and a limited number of NOEs and other constraints for structure calculations. This has resulted in an unprecedented growth in the number of successfully determined NMR structures of large and complex membrane proteins, and this technique now holds great promise for the structure determination of an ever larger body of membrane proteins.
最近的几项进展已将溶液核磁共振光谱法转变为一种具有竞争力、优雅且非常可行的技术，用于在原子分辨率水平上测定膜蛋白的溶液结构。一旦在基于细胞或无细胞的表达和纯化方面达到了合适大小的膜蛋白的良好水平，那么核磁共振提供了多种通用策略的组合，例如，选择合适的氘代或非氘代去污剂、温度和离子强度；用（2）H、（13）C、（15）N进行同位素标记，异亮氨酸（δ1）、亮氨酸和缬氨酸甲基质子有或无质子化；特定氨基酸的组合标记；基于横向弛豫优化核磁共振光谱法、基于非均匀采样的方法以及其他核磁共振实验；使用拉伸聚丙烯酰胺凝胶或DNA纳米管测量残余偶极耦合；以及自旋标记和顺磁弛豫增强。这些进展的策略性组合，再加上配备高场核磁共振光谱仪（高达1 GHz（1）H频率）的高灵敏度低温冷却探头，使坚持不懈的研究人员能够成功克服与核磁共振技术和膜蛋白相关的一些传统陷阱，即灵敏度低、样品稳定性差、光谱拥挤以及用于结构计算的NOE数量有限和其他限制。这导致成功测定的大型和复杂膜蛋白的核磁共振结构数量空前增长，并且该技术现在对于更大数量的膜蛋白的结构测定具有巨大的前景。
