Smith R, Thomas D E, Separovic F, Atkins A R, Cornell B A
Biochemistry Department, University of Queensland, St. Lucia, Australia.
Biophys J. 1989 Aug;56(2):307-14. doi: 10.1016/S0006-3495(89)82677-3.
Solid-state nuclear magnetic resonance (NMR) measurements on 13C-labeled analogues of the ion channel-forming peptide, gramicidin A, have been used to directly determine the structure of this peptide in lipid membranes. Seven gramicidin analogues, each labeled in a single carbonyl group of gly2, L-ala3, D-leu4, L-val7, D-leu10, D-leu12, or D-leu14 were synthesized by the solid-phase method. These gramicidin analogues were incorporated into aligned multilayers of dimyristoylphosphatidylcholine, or diether lipid bearing 14- or 16-carbon chains, at a 1:15 peptide:lipid mole ratio. Proton-enhanced, 13C, solid-state spectra were obtained at several temperatures and over a range of sample orientations with respect to the spectrometer magnetic field to permit accurate measurement of the chemical shift anisotropies. The observed anisotropies indicate that all of the labeled carbonyl bonds are oriented almost parallel to the molecular long axis and perpendicular to the lipid bilayer plane. These orientations are consistent with gramicidin forming a beta 6.3 single-strand helix that is oriented parallel to the methylene chains of the lipid molecules. Comparison of the linewidths from labeled residues that are in the innermost turn of the helix (gly2, ala3, and D-leu4), in the center of the molecule (val7), and in the turn nearest the lipid bilayer surface (D-leu10, D-leu12, and D-leu14) suggests that although the peptide behaves largely as a rigid barrel, segments of the peptide close to the membrane surface possess greater motional freedom. At temperatures above the gel-to-liquid crystalline transition temperature (Tc) the gramicidin molecules rotate, with a less than millisecond correlation time, about the bilayer normal: several degrees below Tc they become immobile on the NMR timescale, without change in the channel conformation. In the L beta' phase the linewidths of the D-leu10, D-leu'2, and D-leu" resonances become equal to those of the other labeled sites, indicating reduced but equivalent motion for all of the peptide carbonyl groups.
对离子通道形成肽短杆菌肽A的13C标记类似物进行的固态核磁共振（NMR）测量已用于直接确定该肽在脂质膜中的结构。通过固相法合成了七种短杆菌肽类似物，每种类似物在gly2、L-ala3、D-leu4、L-val7、D-leu10、D-leu12或D-leu14的单个羰基中进行标记。这些短杆菌肽类似物以1:15的肽:脂质摩尔比掺入二肉豆蔻酰磷脂酰胆碱或带有14或16个碳链的二醚脂质的排列多层膜中。在几个温度下以及相对于光谱仪磁场的一系列样品取向下获得了质子增强的13C固态光谱，以允许准确测量化学位移各向异性。观察到的各向异性表明，所有标记的羰基键几乎都与分子长轴平行且垂直于脂质双层平面。这些取向与短杆菌肽形成与脂质分子的亚甲基链平行取向的β6.3单链螺旋一致。对螺旋最内圈（gly2、ala3和D-leu4）、分子中心（val7）以及最靠近脂质双层表面的圈（D-leu10、D-leu'12和D-leu'14）中标记残基的线宽进行比较表明，尽管该肽在很大程度上表现为刚性桶状，但靠近膜表面的肽段具有更大的运动自由度。在高于凝胶-液晶转变温度（Tc）的温度下，短杆菌肽分子围绕双层法线旋转，相关时间小于一毫秒：在低于Tc几度时，它们在NMR时间尺度上变得不动，通道构象没有变化。在Lβ'相中，D-leu10、D-leu'12和D-leu'共振的线宽变得与其他标记位点的线宽相等，表明所有肽羰基基团的运动减少但等效。
