Partitioning of HIV-1 Gag and Gag-related proteins to membranes
Partitioning of HIV-1 Gag and Gag-related proteins to membranes
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DOI：
10.1021/bi952337x
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发表时间：
1996-04-02
期刊：
影响因子：
2.9
通讯作者：
Carter, C
中科院分区：
文献类型：
--
作者：
Ehrlich, LS;Fong, S;Carter, C
用三种实验系统研究了HIV-1 Gag和Gag相关蛋白与模型膜的结合：（i）大单层磷脂囊泡（LUV）和从大肠杆菌纯化的重组Gag;（ii）加入到哺乳动物细胞提取物中的LUV，其中Gag蛋白通过偶联的转录/翻译系统表达;和（iii）从人红细胞（RBC）纯化的由内而外的质膜囊泡和来自E.杆菌观察到HIV-1 Gag膜相互作用的几个新方面：（i）Gag蛋白以高亲和力与带负电荷表面的模型膜和RBC膜结合。(ii)Gag前体和成熟Gag蛋白的结合对离子强度表现出不同的敏感性，表明前体通过与其任何单个结构域的相互作用定性和定量地不同的相互作用指导膜结合。利用蛋白质中色氨酸残基和插入LUV中的含蒽氧基探针之间的能量转移进行的研究表明，前体蛋白和成熟蛋白在膜表面上的取向是不同的;（iii）Gag寡聚体似乎在高盐条件下促进了高亲和力结合，表明蛋白质-蛋白质相互作用导致形成更强的静电或新的疏水膜结合决定簇。由于与模型膜的结合研究允许定量分析，这些实验方法可以允许识别驱动Gag组装在膜上的相互作用。
The binding of HIV-1 Gag and Gag-related proteins to model membranes was examined using three experimental systems: (i) large unilamellar phospholipid vesicles (LUVs) and recombinant Gag purified from Escherichia coli; (ii) LUVs added to a mammalian cell extract in which Gag proteins were expressed by a coupled transcription/translation system; and (iii) inside-out plasma membrane vesicles purified from human red blood cells (RBC) and recombinant, purified Gag from E. coli. Several novel aspects of HIV-1 Gag membrane interactions were observed: (i) Gag proteins bound with high affinity to both model membranes with a negatively charged surface and to RBC membranes. (ii) Binding of the Gag precursor and mature Gag proteins exhibited different sensitivities to ionic strength indicating that the precursor directed membrane binding through interactions that were qualitatively and quantitatively distinct from those of any of its individual domains. Studies using energy transfer between tryptophan residues in the proteins and anthroyloxy-containing probes inserted In the LUVs indicated that the orientation of the precursor and of the mature proteins on the membrane surface were distinct; (iii) Gag oligomers appear to have facilitated high-affinity binding under high salt conditions, suggesting that protein-protein interactions led to formation of stronger electrostatic or new hydrophobic membrane binding determinants. Since binding studies with model membranes permit quantitative analysis, these experimental approaches may permit identification of interactions that drive Gag assembly on the membrane.