Nicholson L K, Cross T A
Department of Chemistry, Florida State University, Tallahassee 32306-3006.
Biochemistry. 1989 Nov 28;28(24):9379-85. doi: 10.1021/bi00450a019.
Due to the difficulty of obtaining protein/lipid cocrystals for diffraction studies, structural research on intrinsic membrane proteins and polypeptides has been largely restricted to indirect experimental techniques. Hence, many fundamental questions associated with peptide/lipid systems remain unanswered. In particular, the handedness of the gramicidin A transmembrane ion channel incorporated into lipid bilayers has been an open question for nearly two decades. In this study, solid-state 15N NMR spectroscopy is employed to probe directly the secondary structure of the polypeptide backbone. Recent determinations of the 15N chemical shift anisotropy tensor with respect to the molecular frame enable the quantitative evaluation of the 15N chemical shift resonances obtained from oriented dimyristoylphosphatidylcholine (DMPC) bilayer samples containing specific site 15N labeled gramicidin. This direct structural approach verifies the beta-sheet hydrogen-bonding pattern proposed by Urry [Urry, D. W. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 672-676] and determines that in our DMPC bilayer preparations the gramicidin channel is right-handed. Additional structural information is provided by the 15N chemical shift data in the form of orientational constraints on the C alpha-C alpha axis orientation of individual peptides relative to the helix axis. The significance of these solid-state NMR results lies in the direct determination of the helix sense and the verification of the beta-type hydrogen bonding, in the development of the solid-state NMR methods for obtaining such information, and in emphasizing the importance of having direct structural data at atomic resolution.
由于难以获得用于衍射研究的蛋白质/脂质共晶体，对内在膜蛋白和多肽的结构研究在很大程度上局限于间接实验技术。因此，许多与肽/脂质系统相关的基本问题仍未得到解答。特别是，掺入脂质双层中的短杆菌肽A跨膜离子通道的螺旋方向近二十年来一直是个悬而未决的问题。在本研究中，采用固态15N核磁共振光谱直接探测多肽主链的二级结构。最近对相对于分子框架的15N化学位移各向异性张量的测定，使得能够对从含有特定位点15N标记短杆菌肽的定向二肉豆蔻酰磷脂酰胆碱（DMPC）双层样品获得的15N化学位移共振进行定量评估。这种直接的结构方法验证了Urry提出的β-折叠氢键模式[Urry, D. W. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 672 - 676]，并确定在我们的DMPC双层制剂中，短杆菌肽通道是右手螺旋的。15N化学位移数据以单个肽段相对于螺旋轴的Cα - Cα轴取向的取向限制形式提供了额外的结构信息。这些固态核磁共振结果的意义在于直接确定螺旋方向并验证β型氢键，在于开发用于获取此类信息的固态核磁共振方法，以及在于强调获得原子分辨率直接结构数据的重要性。
