Spectral analysis and tryptic susceptibility as probes of HIV-1 capsid protein structure.
Spectral analysis and tryptic susceptibility as probes of HIV-1 capsid protein structure.
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光谱分析和胰蛋白酶敏感性作为 HIV-1 衣壳蛋白结构的探针。
DOI：
10.1006/viro.1994.1565
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发表时间：
1994
期刊：
影响因子：
3.7
通讯作者：
Carter,CA
中科院分区：
文献类型：
--
作者：
Ehrlich,LS;Agresta,BE;Gelfand,CA;Jentoft,J;Carter,CA
使用圆二色性 (CD) 光谱分析比较了从成熟病毒体分离的 HIV-1 衣壳蛋白 (CA, p24) 和从大肠杆菌中表达的重组 Gag-Pol 前体自动加工的 CA 蛋白的结构。获得的完整重组蛋白和病毒蛋白的光谱无法区分，表明由蛋白的一级氨基酸序列指导的主链构型是相似或相同的。一般来说，从 CD 得出的结构预测与提出在几种 RNA 病毒中发现的八链 β 桶基序的模型不一致。然而，该模型的各个方面得到了确定表面暴露区域的实验的支持。生化分析表明重组CA蛋白在体外形成非随机的高阶结构。在生理条件下，蛋白质组装成含有两种包装排列的亚基的寡聚物。在一种布置中，Arg100 附近的中心区域暴露在外，并且在低酶浓度（酶：底物比率 = 1:5000 至 1:100）下容易受到胰蛋白酶消化。此外，在这种排列中，蛋白质容易被双功能剂 DTSSP 交联。另一种排列的蛋白质在低酶浓度下抵抗蛋白水解。这些抗性分子的中心区域无法被识别残基 94 和 107 之间的抗原位点的单特异性抗体接近，并且这些蛋白质不通过 DTSSP 或 EGS 交联。与胰蛋白酶一起孵育后，寡聚物中的抗性分子和源自易感蛋白的片段都迁移为较小的复合物，这表明胰蛋白酶消化的区域稳定了寡聚物单元。结果表明，HIV-1 CA 蛋白的中心区域在高级结构的形成中发挥着作用。此外，Arg100/Gly101 切割产生的 N 端和 C 端部分消化片段的相对稳定性表明，这个暴露的中心区域将蛋白质的两个结构域分开。
The structures of HIV-1 capsid protein (CA, p24) isolated from mature virions and CA protein autoprocessed from a recombinant Gag-Pol precursor expressed inEscherichia coliwere compared using circular dichroic (CD) spectral analysis. The spectra obtained for the intact recombinant and viral proteins were indistinguishable, indicating that the backbone configurations directed by the primary amino acid sequences of the proteins were similar or identical. The structure predictions derived from CD were, in general, inconsistent with a model proposing the eight-stranded beta barrel motif found in several RNA viruses. However, aspects of the model were supported by experiments that identified surface-exposed regions. Biochemical analysis indicated that the recombinant CA protein formed nonrandom higher-ordered structuresin vitro. Under physiological conditions, the protein assembled into oligomers containing subunits in two packing arrangements. In one arrangement, the central region near Arg100 was exposed and susceptible to tryptic digestion at low enzyme concentrations (enzyme:substrate ratios = 1:5000 to 1:100). Also, in this arrangement, the proteins were susceptible to crosslinking by the bifunctional agent DTSSP. Proteins in the other arrangement were resistant to proteolysis at low enzyme concentrations. The central region of these resistant molecules was inaccessible to monospecific antibodies that recognized antigenic sites between residues 94 and 107 and these proteins were not crosslinked by DTSSP or EGS. Following incubation with trypsin, both the resistant molecules and the fragments derived from the susceptible proteins in the oligomer migrated as smaller complexes, suggesting that the regions digested by trypsin stabilized the oligomer unit. The results indicate that the central region of the HIV-1 CA protein plays a role in formation of higher-ordered structures. Moreover, the relative stability of the N- and C-terminal partial digestion fragments arising from cleavage at Arg100/Gly101 suggests that this exposed central region separates two structural domains of the protein.