Kimura S, Naito A, Tuzi S, Saitô H
Department of Life Science, Faculty of Science, Harima Science Garden City, Kouto 3-chome, Kamigori, Hyogo 678-1297, Japan.
Biopolymers. 2001 Jan;58(1):78-88. doi: 10.1002/1097-0282(200101)58:1<78::AID-BIP80>3.0.CO;2-C.
We have recorded (13)C NMR spectra of selectively [3-(13)C]Ala-, [1-(13)C]Ala-, or [1-(13)C]Val-labeled synthetic transmembrane peptides of bacteriorhodopsin (bR) and enzymatically cleaved C-2 fragment in the solid and dimyristoylphosphatidylcholine bilayer. It turned out that these transmembrane peptides either in hexafluoroisopropanol or cast from it take an ordinary alpha-helix (alpha(I)-helix) irrespective of their amino acid sequences with reference to the conformation-dependent (13)C chemical shifts of (Ala)(n) taking the alpha-helix form. These transmembrane peptides are not always static in the lipid bilayer as in the solid state but undergo rigid-body motions with various frequencies as estimated from suppressed peaks either by fast isotropic or large-amplitude motions (>10(8) Hz) or intermediate frequencies (10(5) or 10(3) Hz). Further, (13)C chemical shifts of the [3-(13)C]Ala-labeled peptides in the bilayer were displaced downfield by 0.3-1.1 ppm depending upon amino acid sequence with respect to those in the solid state, which were explained in terms of local conformational fluctuation (10(2) Hz) deviated from the torsion angles (alpha(II)-helix) from those of standard alpha-helix, under anisotropic environment in lipid bilayer, in addition to the above-mentioned rigid-body motions. The carbonyl (13)C peaks, on the other hand, are not sensitively displaced by such local anisotropic fluctuations, because they are more sensitive to the manner of hydrogen-bond interactions. The amino acid sequences of these peptides inserted within the bilayer were not always the same as those of intact bR, causing disposition of the transmembrane alpha-helical segment from that of intact bR. Finally, we confirmed that the (13)C NMR peak positions of the random coil form are located at the boundary between the alpha-helix and a turned structure in loop regions.
我们记录了在固体和二肉豆蔻酰磷脂酰胆碱双层中，选择性地用[3-(13)C]丙氨酸、[1-(13)C]丙氨酸或[1-(13)C]缬氨酸标记的细菌视紫红质（bR）的合成跨膜肽以及酶切的C-2片段的(13)C NMR光谱。结果表明，这些跨膜肽无论是在六氟异丙醇中还是从中浇铸出来，都呈现出普通的α-螺旋（α(I)-螺旋），这与它们的氨基酸序列无关，参考呈α-螺旋形式的(Ala)(n)的构象依赖性(13)C化学位移。这些跨膜肽在脂质双层中并不总是像在固态中那样静止不动，而是会进行刚体运动，其频率各异，这是根据快速各向同性或大幅度运动（>10(8)Hz）或中频（10(5)或10(3)Hz）导致的峰抑制来估计的。此外，双层中[3-(13)C]丙氨酸标记的肽的(13)C化学位移相对于固态中的位移向低场移动了0.3 - 1.1 ppm，这取决于氨基酸序列，除了上述刚体运动外，这可以用脂质双层各向异性环境中偏离标准α-螺旋扭转角（α(II)-螺旋）的局部构象波动（10(2)Hz）来解释。另一方面，羰基(13)C峰对这种局部各向异性波动不敏感，因为它们对氢键相互作用的方式更敏感。插入双层内的这些肽的氨基酸序列并不总是与完整bR的相同，导致跨膜α-螺旋片段的排列与完整bR的不同。最后，我们证实了无规卷曲形式的(13)C NMR峰位置位于α-螺旋和环区域中转折结构之间的边界处。
