Chao S F, Chan V L, Juranka P, Kaplan A H, Swanstrom R, Hutchison C A
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill 27599, USA.
Nucleic Acids Res. 1995 Mar 11;23(5):803-10. doi: 10.1093/nar/23.5.803.
We have analyzed 154 single amino acid replacement mutants within a 40 amino acid region (residues 164-203) of the reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1). This region consists of two antiparallel beta-strands (strands 9 and 10) flanked by two alpha helices (E and F). The structure of this region of the 'palm' subdomain is conserved in a variety of DNA and RNA polymerases, indicating a critical role in enzyme structure and function. Functional assays were performed by screening RT activity of mutants expressed in E. coli. A functionally important region corresponding closely to beta-strands 9 and 10 and the loop joining them was revealed by its mutational sensitivity. Structural analysis of mutants was performed by using Western blots to assay correct folding, which is required for processing to produce the mature p66 and p51 RT species. This analysis indicates that beta-strand 10 is a structurally important region. Combined analysis of these two assays revealed diagnostic patterns of mutational sensitivity which identify key positions in the RT sequence at which a specific amino acid side chain is critical, either for structure or function, as well as residues which are external to the RT structure. This work illustrates the utility of large-scale mutagenesis in relating primary sequence to significant features of protein structure and function.
我们分析了来自人类免疫缺陷病毒1型（HIV-1）逆转录酶（RT）40个氨基酸区域（第164 - 203位氨基酸）内的154个单氨基酸替换突变体。该区域由两条反平行的β链（链9和链10）组成，两侧为两条α螺旋（E和F）。“手掌”亚结构域的这一区域结构在多种DNA和RNA聚合酶中保守，表明其在酶的结构和功能中起关键作用。通过筛选在大肠杆菌中表达的突变体的RT活性进行功能测定。通过其突变敏感性揭示了一个与β链9和10以及连接它们的环紧密对应的功能重要区域。通过使用蛋白质免疫印迹法检测正确折叠来进行突变体的结构分析，这是产生成熟的p66和p51 RT物种所必需的。该分析表明β链10是一个结构重要区域。这两种测定的联合分析揭示了突变敏感性的诊断模式，其确定了RT序列中的关键位置，在这些位置特定氨基酸侧链对于结构或功能至关重要，以及RT结构外部的残基。这项工作说明了大规模诱变在将一级序列与蛋白质结构和功能的重要特征相关联方面的实用性。
