N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange
N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange
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DOI：
10.1021/bi962252b
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发表时间：
1997-04-15
期刊：
影响因子：
2.9
通讯作者：
Chabre, M
中科院分区：
文献类型：
--
作者：
Antonny, B;BeraudDufour, S;Chabre, M
GDP/GTP交换调节小G蛋白ADP-核糖化因子-1与膜脂的相互作用：如果ARF(GDP)主要是可溶的，则ARF(GTP)与脂泡紧密结合。以前的研究表明，这种依赖GTP的结合在去除N-末端肉豆蔻酸酯后持续存在，但在17个N-末端残基进一步缺失后被取消。这提示了这种两亲性多肽在脂膜结合中的作用。在ARF(GDP)晶体结构中，2-13肽是螺旋状的，其疏水残基埋在蛋白质核心中。当ARF转换到GTP状态时，这些残基可能会插入膜脂中。我们研究了ARF与特定成分的单层囊泡的结合。ArF(GDP)通过肉豆蔻酸盐的疏水相互作用和阳离子残基与阴离子脂类的静电相互作用与囊泡弱结合。磷脂酰肌醇4，5-双(磷酸盐)除了严格的静电作用外，没有其他特殊的作用。通过荧光能量转移，通过标记的脂泡上的ArF(GTP-S)解离速率来评估Arf(GTP)与脂质相互作用的强度。ARF(GTP-γS)从中性PC囊泡缓慢解离(tau(OFF)x 75 S)。包括30%的阴离子磷脂只增加了3倍的tau(关)。通过点突变降低N端肽的疏水性有更大的影响：F9A和L8A-F9A取代分别加速ARF(GTP Gamma S)从囊泡中解离7倍和100倍。这有力地表明，在GDP/GTP交换时，N-末端螺旋从蛋白质核心释放，因此其疏水残基可以与膜磷脂相互作用。
GDP/GTP exchange modulates the interaction of the small G-protein ADP-ribosylation factor-1 with membrane lipids: if ARF(GDP) is mostly soluble, ARF(GTP) binds tightly to lipid vesicles. Previous studies have shown that this GTP-dependent binding persists upon removal of the N-terminal myristate but is abolished following further deletion of the 17 N-terminal residues. This suggests a role for this amphipathic peptide in lipid membrane binding. In the ARF(GDP) crystal structure, the 2-13 peptide is helical, with its hydrophobic residues buried in the protein core. When ARF switches to the GTP state, these residues may insert into membrane lipids. We have studied the binding of ARF to model unilamellar vesicles of defined composition. ARF(GDP) binds weakly to vesicles through hydrophobic interaction of the myristate and electrostatic interaction of cationic residues with anionic lipids. Phosphatidylinositol 4,5-bis(phosphate) shows no specific effects other than strictly electrostatic. By using fluorescence energy transfer, the strength of the ARF(GTP)-lipid interaction is assessed via the dissociation rate of ARF(GTP gamma s) from labeled lipid vesicles. ARF(GTP gamma S) dissociates slowly (tau(off) x 75 s) from neutral PC vesicles. Including 30% anionic phospholipids increases tau(off) by only 3-fold. Reducing the N-terminal peptide hydrophobicity by point mutations had larger effects: F9A and L8A-F9A substitutions accelerate the dissociation of ARF(GTP gamma S) from vesicles by factors of 7 and 100, respectively. This strongly suggests that, upon GDP/GTP exchange, the N-terminal helix is released from the protein core so its hydrophobic residues can interact with membrane phospholipids.