Department of Chemistry, Iowa State University, Ames, Iowa 50011, USA.
J Phys Chem B. 2011 Sep 15;115(36):10758-67. doi: 10.1021/jp205002n. Epub 2011 Aug 18.
A challenge in the application of solid-state NMR spectroscopy to membrane peptides and proteins is the relatively broad line widths compared to those for solution NMR spectra. To understand the linewidth contributions to membrane protein NMR spectra, we have measured the inhomogeneous and homogeneous line widths of several well-studied membrane peptides under immobilized conditions. (13)C T(2) relaxation times of uniformly (13)C-labeled residues show that the homogeneous line widths of the peptides are comparable to those of crystalline model compounds under identical (1)H decoupling and magic angle spinning conditions, indicating that the homogeneous line widths are determined by conformation-independent factors, including residual dipolar coupling, J-coupling, and intrinsic T(2) relaxation. However, the membrane peptides exhibit larger apparent line widths than the crystalline compounds, indicating conformational disorder. A cationic cell-penetrating peptide, the human immunodeficiency virus TAT, exhibits the largest apparent line widths, which are about five-fold larger than the homogeneous line widths, while the transmembrane helix of the influenza M2 peptide and the β-hairpin antimicrobial peptide PG-1 show moderately larger apparent line widths than the crystalline compounds. These results are consistent with the random coil nature of the TAT peptide, which contrasts with the intramolecularly hydrogen bonded M2 and PG-1. Cross peak line shapes of 2D double-quantum correlation spectra show that the conformational disorder can occur at the residue level and can result from three origins, lipid-peptide interaction, intrinsic conformational disorder encoded in the amino acid sequence, and side-chain rotameric averaging. A particularly important lipid-peptide interaction for cationic membrane peptides is guanidinium-phosphate ion pair interaction. Thus, NMR line widths and line shapes are useful for understanding the conformational disorder of membrane peptides and proteins.
固态 NMR 光谱学在膜肽和蛋白质应用中的一个挑战是与溶液 NMR 谱相比，谱线较宽。为了了解膜蛋白 NMR 谱线宽度的贡献，我们在固定化条件下测量了几种经过充分研究的膜肽的不均匀和均匀线宽。均匀（13）C 标记残基的（13）C T（2）弛豫时间表明，在相同的（1）H 去耦和魔角旋转条件下，肽的均匀线宽与结晶模型化合物相当，表明均匀线宽由构象独立的因素决定，包括残余偶极耦合、J 耦合和固有 T（2）弛豫。然而，膜肽表现出比结晶化合物更大的表观线宽，表明构象无序。阳离子细胞穿透肽，人类免疫缺陷病毒 TAT，表现出最大的表观线宽，比均匀线宽大约大五倍，而流感 M2 肽的跨膜螺旋和β发夹抗菌肽 PG-1 表现出比结晶化合物稍大的表观线宽。这些结果与 TAT 肽的无规卷曲性质一致，与分子内氢键形成的 M2 和 PG-1 形成对比。2D 双量子相关谱的交叉峰线形状表明，构象无序可以在残基水平上发生，并可能源于三个来源，即脂质-肽相互作用、氨基酸序列中编码的固有构象无序和侧链旋转异构体平均化。对于阳离子膜肽，特别重要的脂质-肽相互作用是胍-磷酸离子对相互作用。因此，NMR 线宽和线形状有助于理解膜肽和蛋白质的构象无序。
