Institute of Molecular Biophysics, Department of Chemistry and Biochemistry, and National High Magnetic Field Laboratory, Florida State University , Tallahassee, Florida 32310, United States.
Acc Chem Res. 2013 Sep 17;46(9):2172-81. doi: 10.1021/ar3003442. Epub 2013 Mar 7.
Unlike water soluble proteins, the structures of helical transmembrane proteins depend on a very complex environment. These proteins sit in the midst of dramatic electrical and chemical gradients and are often subject to variations in the lateral pressure profile, order parameters, dielectric constant, and other properties. Solid state NMR is a collection of tools that can characterize high resolution membrane protein structure in this environment. Indeed, prior work has shown that this complex environment significantly influences transmembrane protein structure. Therefore, it is important to characterize such structures under conditions that closely resemble its native environment. Researchers have used two approaches to gain protein structural restraints via solid state NMR spectroscopy. The more traditional approach uses magic angle sample spinning to generate isotropic chemical shifts, much like solution NMR. As with solution NMR, researchers can analyze the backbone chemical shifts to obtain torsional restraints. They can also examine nuclear spin interactions between nearby atoms to obtain distances between atomic sites. Unfortunately, for membrane proteins in lipid preparations, the spectral resolution is not adequate to obtain complete resonance assignments. Researchers have developed another approach for gaining structural restraints from membrane proteins: the use of uniformly oriented lipid bilayers, which provides a method for obtaining high resolution orientational restraints. When the bilayers are aligned with respect to the magnetic field of the NMR spectrometer, researchers can obtain orientational restraints in which atomic sites in the protein are restrained relative to the alignment axis. However, this approach does not allow researchers to determine the relative packing between helices. By combining the two approaches, we can take advantage of the information acquired from each technique to minimize the challenges and maximize the quality of the structural results. By combining the distance, torsional, and orientational restraints, we can characterize high resolution membrane protein structure in native-like lipid bilayer environments.
与水溶性蛋白质不同，螺旋跨膜蛋白质的结构取决于非常复杂的环境。这些蛋白质位于剧烈的电和化学梯度中间，并且经常受到侧向压力分布、有序参数、介电常数和其他性质变化的影响。固态 NMR 是一组工具，可以在这种环境中对高分辨率膜蛋白结构进行特征化。事实上，先前的工作表明，这种复杂的环境会显著影响跨膜蛋白质的结构。因此，在非常接近其天然环境的条件下对这种结构进行特征化是很重要的。
研究人员已经使用了两种方法通过固态 NMR 光谱学获得蛋白质结构约束。更传统的方法是使用魔角旋转样品（MAS）来产生各向同性化学位移，这与溶液 NMR 非常相似。与溶液 NMR 一样，研究人员可以分析骨架化学位移以获得扭转约束。他们还可以检查相邻原子之间的核自旋相互作用，以获得原子位置之间的距离。
不幸的是，对于脂质制剂中的膜蛋白，谱分辨率不足以获得完整的共振分配。研究人员已经开发出另一种从膜蛋白中获得结构约束的方法：使用均匀取向的脂质双层，这提供了一种获得高分辨率取向约束的方法。当双层相对于 NMR 光谱仪的磁场排列时，研究人员可以获得原子位置相对于排列轴的取向约束。然而，这种方法不允许研究人员确定螺旋之间的相对堆积。通过结合这两种方法，我们可以利用从每种技术中获得的信息来最小化挑战并最大化结构结果的质量。通过结合距离、扭转和取向约束，我们可以在类似天然的脂质双层环境中对高分辨率膜蛋白结构进行特征化。
