Conformation of the gramicidin A channel in phospholipid vesicles: a fluorine-19 nuclear magnetic resonance study.
Conformation of the gramicidin A channel in phospholipid vesicles: a fluorine-19 nuclear magnetic resonance study.
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磷脂囊泡中短杆菌肽 A 通道的构象：氟 19 核磁共振研究。
DOI：
10.1021/bi00337a019
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Blout,ER
中科院分区：
文献类型：
--
作者：
Weinstein,S;Durkin,JT;Veatch,WR;Blout,ER
哈佛医学院生物化学和药理学系，波士顿，马萨诸塞州 02115 收到 1985 年 1 月 3 日摘要：19F NMR 显示肽离子载体短杆菌肽 A 的膜构象可通过 Urry 提出的 N 端至 N 端/3LD 螺旋二聚体模型进行描述 [Urry, DW (1971) Proc.国家。阿卡德。科学。我们。 A. 68, 672-676]，合成制备了 N 端和 C 端均带有 19F 标记的短杆菌肽的完全活性类似物。标记的肽被掺入二肉豆蔻酰磷脂酰胆碱的小单层囊泡中。对水性或亲脂性顺磁探针的标记可及性的测量表明，短杆菌肽的 N 末端位于膜内部，C 末端位于膜表面。在针对短杆菌肽结构提出的具体模型中，这些数据仅与 Urry 的数据一致。 C-末端 19F NMR 峰 id 囊泡实际上由三个重叠峰组成。使用水转移试剂 Tm3+ 的实验表明，囊泡内部和外部小叶中的 C 端 19F 核以不同频率共振。外叶峰依次由两个重叠峰组成，可能是由于 C 端标记的局部重排。（短杆菌肽 A 是一种线性十五肽，形成单价阳离子特异性的跨膜通道。[有关最近的评论，请参见 Andersen (1984)]。其结构的简单性使其成为一个实验上易于理解的系统，用于了解离子跨生物膜转运的分子基础。此类研究的首要要求是确定短杆菌肽通道。短杆菌肽由交替的 d-和 L-氨基酸组成，其 N-末端和 C-末端分别被甲酰基和乙醇胺基团封闭。主要形式[Val]短杆菌肽 A 的氨基酸序列为。 HCO-L-Val1-Gly2-L-Ala3-D-Leu4-L-Ala5-D-Val6-L-Val7-D-Val8-L-Trpg-D-Leu，0-L-Trpn-D-Leu12-L-Trp13-D-Leu14-L-Trp15-NHCH2CH2OH（Sarges & Witkop， 1 短杆菌肽传导通道是二聚体（Tostesonet al., 1968；Goodall, 1970；Urry et al., 1971；Kolb et al., 1975；Veatch et al., 1975；Bamberg & Janko, 1977；Veatch & Stryer, 1977）。根据化学修饰的短杆菌肽的电导研究和光谱数据，模型 A 和 B 是膜内部首尾相连的单螺旋通道，模型 C 和 D 是双螺旋，其中两个肽链都跨越膜，模型 A 是 Urry 首次提出的 N 端到 N 端 0LD 螺旋二聚体。 （1971；Urry 等人，1971；然后称为
Departments of Biological Chemistry and Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 Received January 3, 1985 abstract: The membrane conformation of the peptide ionophore gramicidin A is shown by 19F NMR to be described by the N-terminal to N-terminal/3LD helical dimer model proposed by Urry [Urry, DW (1971) Proc. Natl. Acad. Sci. US. A. 68, 672-676], Fully activeanalogues of gramicidin with 19F labels at both the N-and C-termini are prepared synthetically. Labeled peptides are incorporated into small unilamellar vesicles of dimyristoylphosphatidylcholine. Measurements of the accessibility of the labels to either aqueous or lipophilic paramagnetic probes show that the N-terminus of gramicidin is located in the membrane interior and the C-terminus is at the membrane surface. Of the specific models proposed for the structure of gramicidin, these data are consistent only with that of Urry. The C-terminal 19F NMR peak id vesicles actually consists of three overlapping peaks. Experiments with the aqueous shift reagent Tm3+ show that C-terminal 19F nuclei in the inner and in the outer leaflets of vesicles resonate at different frequencies. The outer leaflet peak in turn consists of two overlapping peaks, possibly due to a local rearrangement of the C-terminal label.(gramicidin A is a linear pentadecapeptide that forms transmembrane channels specific for monovalent cations.[For a recent review, see Andersen (1984)]. The simplicityof its structure makes it an experimentally approachablesystem for understanding the molecular basis for ion transport across biological membranes. The first requirement for such studies is determination of the conformation of the gramicidin channel. Gramicidin is composed of alternating d-and L-amino acids, and its N-and C-termini are blocked by a formyl and ethanolamine group, respectively. The amino acid sequence of the major form,[Val] gramicidin A, is HCO-L-Val1-Gly2-L-Ala3-D-Leu4-L-Ala5-D-Val6-L-Val7-D-Val8-L-Trpg-D-Leu, 0-L-Trpn-D-Leu12-L-Trp13-D-Leu14-L-Trp15-NHCH2CH2OH (Sarges & Witkop, 1965a). 1 The conducting gramicidin channel is a dimer (Tostesonet al., 1968; Goodall, 1970; Urry et al., 1971; Kolb et al., 1975; Veatch et al., 1975; Bamberg & Janko, 1977; Veatch & Stryer, 1977). Two classes of models have been proposed for the conformationof gramicidin in solution and in the membrane, on the basis of conductance studies of chemically modified gramicidins and on the basis of spectroscopic data. These are shown schematically in Figure1. Models A and B are single-helical channels dimerized end to end in the membrane interior. Models Cand D are double helices in which both peptide chains span the membrane. Model A is the N-terminal to N-terminal 0LD helical dimer first proposed by Urry (1971; Urry et al., 1971; then called