Bilayer interactions of indolicidin, a small antimicrobial peptide rich in tryptophan, proline, and basic amino acids
Bilayer interactions of indolicidin, a small antimicrobial peptide rich in tryptophan, proline, and basic amino acids
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DOI：
10.1016/s0006-3495(97)78713-7
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发表时间：
1997-02-01
影响因子：
3.4
通讯作者：
White, SH
中科院分区：
文献类型：
--
作者：
Ladokhin, AS;Selsted, ME;White, SH
色氨酸、脯氨酸和碱性氨基酸都被认为在膜蛋白的组装和结构中起重要作用。Indolicidin是一种从牛中性粒细胞的细胞质颗粒中分离的抗菌13-残基肽酰胺，高度富集这些氨基酸：5个色氨酸，3个脯氨酸，3个碱性残基，没有酸性残基。与这些氨基酸在膜蛋白组装中可能的重要性一致，已知indolicidin具有高度膜活性，并且据信通过破坏细胞膜起作用。因此，我们已经研究了天然indolicidin与大的单层囊泡（LUV）形成的palmitoyloleoyt磷脂酰胆碱（POPC），和palmitoyloleoyl磷脂酰甘油（POPG）的相互作用，为了使用它作为一个模型系统，用于研究膜蛋白插入和评估的相对贡献的疏水和静电力的肽-双层相互作用。平衡透析测量表明indolicidin与中性POPC囊泡和阴离子POPG囊泡两者均强烈但可逆地结合，转移自由能分别为-8.8 +/-0.2和-11.5 +/-0.4千卡/摩尔。POPG囊泡的极强的分区有必要开发一种新的平衡透析方法，详细描述。色氨酸荧光测量表明，indolicidin是位于双层界面和吲哚荧光的影响所使用的脂质的类型形成的LUV。圆二色性（CD）的测量显示在水溶液和散装有机溶液中的无序构象和有点有序，但不是α-螺旋，构象在SDS胶束和脂质双层。荧光再激发测量（Ladokhin等人，1995. Biophys. J. 69：1964-1971）在装载有荧光团/猝灭剂对8-氨基萘-1，3，6三磺酸（ANTS）/对二甲苯-双-溴化吡啶（DPX）的囊泡上的研究表明，吲哚菌素诱导膜透化。对于阴离子POPG，泄漏分级优先考虑阳离子DPX的释放，而不是阴离子ANTS。对于中性POPC囊泡，没有观察到这种偏好。通过附录中开发的三种定量指标判断，POPG囊泡的泄漏诱导比POPC囊泡更有效。
Tryptophan, proline, and basic amino acids have all been implicated as being important in the assembly and structure of membrane proteins. Indolicidin, an antimicrobial 13-residue peptide-amide isolated from the cytoplasmic granules of bovine neutrophils, is highly enriched in these amino acids: five tryptophans, three prolines, three basic residues, and no acidic residues. Consistent with the likely importance of these amino acids in membrane protein assembly, indolicidin is known to be highly membrane-active and is believed to act by disruption of cell membranes. We have, therefore, examined the interactions of native indolicidin with large unilamellar Vesicles (LUV) formed from palmitoyloleoytphosphatidylcholine (POPC), and palmitoyloleoylphosphatidylglycerol (POPG), in order to use it as a model system for studying membrane protein insertion and for evaluating the relative contributions of hydrophobic and electrostatic forces in peptide-bilayer interactions. Equilibrium dialysis measurements indicate that indolicidin binds strongly, but reversibly, to both neutral POPC and anionic POPG vesicles with free energies of transfer of -8.8 +/- 0.2 and -11.5 +/- 0.4 kcal/mol, respectively. The extremely strong partitioning into POPG vesicles necessitated the development of a new equilibrium dialysis method that is described in detail. Tryptophan fluorescence measurements show that indolicidin is located in the bilayer interface and that indole fluorescence is affected by the type of lipid used to form the LUVs. Circular dichroism (CD) measurements reveal unordered conformations in aqueous and bulk organic solutions and a somewhat more ordered, but not alpha-helical, conformation in SDS micelles and lipid bilayers. Fluorescence requenching measurements (Ladokhin et al. 1995. Biophys. J. 69:1964-1971) on vesicles loaded with the fluorophore/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX), show that indolicidin induces membrane permeabilization. For anionic POPG, leakage is graded with a high preference for the release of cationic DPX over anionic ANTS. For neutral POPC vesicles no such preference is observed. Leakage induction is more effective with POPG vesicles than with POPC vesicles, as judged by three quantitative measures that are developed in the Appendix.