Institute for Biophysical Chemistry and Centre for Biomolecular Magnetic Resonance, Goethe University Frankfurt am Main , Max von Laue Str. 9, 60438 Frankfurt am Main, Germany.
Acc Chem Res. 2013 Sep 17;46(9):2164-71. doi: 10.1021/ar4000289. Epub 2013 Jun 7.
Membrane proteins catalyze reactions at the cell membrane and facilitate thetransport of molecules or signals across the membrane. Recently researchers have made great progress in understanding the structural biology of membrane proteins, mainly based on X-ray crystallography. In addition, the application of complementary spectroscopic techniques has allowed researchers to develop a functional understanding of these proteins. Solid-state NMR has become an indispensable tool for the structure-function analysis of insoluble proteins and protein complexes. It offers the possibility of investigating membrane proteins directly in their environment, which provides essential information about the intrinsic coupling of protein structure and functional dynamics within the lipid bilayer. However, to date, researchers have hardly explored the enzymology of mem-brane proteins. In this Account, we review the perspectives for investigating membrane-bound enzymes by solid-state NMR. Understanding enzyme mechanisms requires access to kinetic parameters, structural analysis of the catalytic center, knowledge of the 3D structure and methods to follow the structural dynamics of the enzyme during the catalytic cycle. In principle, solid-state NMR can address all of these issues. Researchers can characterize the enzyme kinetics by observing substrate turnover within the membrane or at the membrane interphase in a time-resolved fashion as shown for diacylglycerol kinase. Solid-state NMR has also provided a mechanistic understanding of soluble enzymes including triosephosphate isomerase (TIM) and different metal-binding proteins, which demonstrates a promising perspective also for membrane proteins. The increasing availability of high magnetic fields and the development of new experimental schemes and computational protocols have made it easier to determine 3D structure using solid-state NMR. Dynamic nuclear polarization, a key technique to boost sensitivity of solid-state NMR at low temperatures, can help with the analysis of thermally trapped catalytic intermediates, while methods to improve signal-to-noise per time unit enable the real-time measurement of kinetics of conformational changes during the catalytic cycle.
膜蛋白在细胞膜上催化反应，并促进分子或信号跨膜运输。最近，研究人员在理解膜蛋白的结构生物学方面取得了重大进展，主要基于 X 射线晶体学。此外，互补光谱技术的应用使研究人员能够对这些蛋白质进行功能理解。固态 NMR 已成为分析不溶性蛋白质和蛋白质复合物结构-功能的不可或缺的工具。它提供了在其环境中直接研究膜蛋白的可能性，这为了解蛋白质结构和功能动力学内在偶联提供了必要的信息，这种偶联存在于脂质双层内。然而，迄今为止，研究人员几乎没有探索过膜蛋白的酶学。在本综述中，我们回顾了通过固态 NMR 研究膜结合酶的前景。理解酶机制需要获得动力学参数、催化中心的结构分析、3D 结构的知识以及在催化循环中跟踪酶结构动力学的方法。原则上，固态 NMR 可以解决所有这些问题。研究人员可以通过在膜内或在膜相间以时间分辨的方式观察底物周转来表征酶动力学，如二酰基甘油激酶所示。固态 NMR 还提供了对可溶性酶的机制理解，包括磷酸丙糖异构酶 (TIM) 和不同的金属结合蛋白，这也为膜蛋白展示了一个很有前途的前景。高磁场的日益普及以及新实验方案和计算协议的发展，使得使用固态 NMR 确定 3D 结构变得更加容易。固态 NMR 低温下提高灵敏度的关键技术动态核极化，有助于分析热捕获的催化中间体，而提高信噪比的方法则使在催化循环期间进行构象变化动力学的实时测量成为可能。
