Design of amphiphilic protein maquettes: Controlling assembly, membrane insertion, and cofactor interactions
Design of amphiphilic protein maquettes: Controlling assembly, membrane insertion, and cofactor interactions
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DOI：
10.1021/bi050695m
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发表时间：
2005-09-20
期刊：
影响因子：
2.9
通讯作者：
Dutton, PL
中科院分区：
文献类型：
--
作者：
Discher, BM;Noy, D;Dutton, PL
我们已经设计了结合选定的亲脂性（LP）和亲水性（HP）序列，组装成两亲性（AP）的α-螺旋束复制天然膜蛋白的关键结构特征和功能元件的多肽。这里开发的主要AP模型（API）通过柔性连接序列（GGNG）将来自结构化二血红素-四-α-螺旋束（HP 1）的血红素结合序列的14个残基与来自流感病毒的天然四-α-螺旋M2通道的跨膜LP结构域的24个残基连接，以制备42个氨基酸的肽。单个API螺旋（没有连接环）在洗涤剂中组装成四个α-螺旋束，如通过分析性超离心所观察到的。螺旋平行取向，如相邻血红素的典型相互作用所示。AP 1在非极性-极性界面上矢量定向，并且容易以>97%的效率并入磷脂囊泡中，尽管最可能没有矢量偏差。单和二血红素-AP 1在膜增强功能元件以及建立在相关的HP类似物。这些包括血红素与内部谷氨酸盐的强氧化还原电荷耦合和内部电场效应，引起相邻血红素的氧化还原电位的显著160 mV分裂，从而导致血红素结合亲和力的差异。AP模型变体AP 2和AP 3通过从呼吸细胞色素bc的跨膜d-螺旋中选择B（H）血红素结合序列，从HP结构域中去除了血红素连接组氨酸，并在LP结构域中包含血红素连接组氨酸（1）。这些代表了AP maquettes的第一个例子，血红素和细菌叶绿素结合位点位于LP结构域内。
We have designed polypeptides combining selected lipophilic (LP) and hydrophilic (HP) sequences that assemble into amphiphilic (AP) alpha-helical bundles to reproduce key structure characteristics and functional elements of natural membrane proteins. The principal AP maquette (API) developed here joins 14 residues of a heme binding sequence from a structured diheme-four-alpha-helical bundle (HP1), with 24 residues of a membrane-spanning LP domain from the natural four-alpha-helical M2 channel of the influenza virus, through a flexible linking sequence (GGNG) to make a 42 amino acid peptide. The individual API helices (without connecting loops) assemble in detergent into four-a-helical bundles as observed by analytical ultracentrifugation. The helices are oriented parallel as indicated by interactions typical of adjacent hemes. AP1 orients vectorially at nonpolar-polar interfaces and readily incorporates into phospholipid vesicles with >97% efficiency, although most probably without vectorial bias. Mono- and diheme-AP1 in membranes enhance functional elements well established in related HP analogues. These include strong redox charge coupling of heme with interior glutamates and internal electric field effects eliciting a remarkable 160 mV splitting of the redox potentials of adjacent hemes that leads to differential heme binding affinities. The AP maquette variants, AP2 and AP3, removed heme-ligating histidines from the HP domain and included heme-ligating histidines in LP domains by selecting the b(H) heme binding sequence from the membrane-spanning d-helix of respiratory cytochrome bc(1). These represent the first examples of AP maquettes with heme and bacteriochlorophyll binding sites located within the LP domains.