Ketchem R, Roux B, Cross T
Center for Interdisciplinary Magnetic Resonance at the National High Magnetic Field Laboratory, Florida State University, Tallahassee 32306-4005, USA.
Structure. 1997 Dec 15;5(12):1655-69. doi: 10.1016/s0969-2126(97)00312-2.
Solid-state nuclear magnetic resonance (NMR) spectroscopy provides novel structural constraints from uniformly aligned samples. These orientational constraints orient specific atomic sites with respect to the magnetic field direction and the unique molecular axis of alignment. Solid-state NMR is uniquely and ideally suited for providing such structural constraints on polypeptides and proteins in a lamellar phase lipid environment. Membrane protein structure represents a great challenge for structural biologists; a new approach for characterizing high resolution three-dimensional structure in such an environment is needed.
The optimal use of orientational constraints for defining three-dimensional structures is demonstrated with the elucidation of the gramicidin A channel structure at high resolution. Initial structures are refined against both the experimental constraints and the CHARMM energy using a novel simulated-annealing protocol to define torsion angle solutions with an error bar of approximately +/- 5 degrees.
This analysis results in the determination of a high-resolution, time averaged structure of gramicidin A obtained in a lipid bilayer environment above the gel-to-liquid crystalline phase transition temperature. It is demonstrated that solid-state NMR can be used to establish polypeptide, and potentially protein, structures in such an environment. Furthermore, this high-resolution structure is demonstrated to provide new insights into polypeptide function. For the gramicidin A channel the roles of the indole groups that facilitate ion transport and details of the cation solvation environment provided by the amide oxygens are characterized.
固态核磁共振（NMR）光谱学可从均匀排列的样品中提供新的结构限制。这些取向限制使特定原子位点相对于磁场方向和独特的分子排列轴定向。固态NMR特别适合且理想地适用于在层状相脂质环境中为多肽和蛋白质提供此类结构限制。膜蛋白结构对结构生物学家来说是一个巨大的挑战；需要一种新方法来表征这种环境中的高分辨率三维结构。
通过高分辨率阐明短杆菌肽A通道结构，证明了取向限制在定义三维结构中的最佳应用。使用一种新颖的模拟退火协议，针对实验限制和CHARMM能量对初始结构进行优化，以定义误差范围约为±5度的扭转角解。
该分析确定了在高于凝胶 - 液晶相转变温度的脂质双层环境中获得的短杆菌肽A的高分辨率、时间平均结构。结果表明，固态NMR可用于在此类环境中建立多肽以及潜在的蛋白质结构。此外，该高分辨率结构为多肽功能提供了新的见解。对于短杆菌肽A通道，表征了促进离子运输的吲哚基团的作用以及酰胺氧提供的阳离子溶剂化环境的细节。
