Wrobel J A, Chao S F, Conrad M J, Merker J D, Swanstrom R, Pielak G J, Hutchison C A
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599, USA.
Proc Natl Acad Sci U S A. 1998 Jan 20;95(2):638-45. doi: 10.1073/pnas.95.2.638.
By using oligonucleotide-directed saturation mutagenesis, we collected 366 different single amino acid substitutions in a 109-aa segment (residues 95-203) in the fingers and palm subdomains of the HIV-1 reverse transcriptase (RT), the enzyme that replicates the viral genome. After expression in Escherichia coli, two phenotypic assays were performed. The first assay tested for RNA-dependent DNA polymerase activity. The other assay used Western blot analysis to estimate the stability of each mutant protein by measuring the processing of the RT into its mature heterodimeric form, consisting of a 66-kDa subunit and a 51-kDa subunit. The resulting phenotypic data provided a "genetic" means to identify amino acid side chains that are important for protein function or stability, as well as side chains located on the protein surface. Several HIV-1 RT crystal structures were used to evaluate the mutational analysis. Our genetic map correlates well with the crystal structures. Combining our phenotype data with crystallographic data allowed us to study the genetically defined critical residues. The important functional residues are found near the enzyme active site. Many residues important for the stability of the RT participate in potential hydrogen bonding or hydrophobic interactions in the protein interior. In addition to providing a better understanding of the HIV-1 RT, this work demonstrates the utility of saturation mutagenesis to study the function, structure, and stability of proteins in general. This strategy should be useful for studying proteins for which no crystallographic data are available.
通过使用寡核苷酸定向饱和诱变，我们在HIV-1逆转录酶（RT）的指状和手掌亚结构域的109个氨基酸片段（第95-203位残基）中收集了366种不同的单氨基酸取代，该酶负责复制病毒基因组。在大肠杆菌中表达后，进行了两种表型分析。第一种分析检测RNA依赖性DNA聚合酶活性。另一种分析使用蛋白质印迹分析，通过测量RT加工成其成熟的异二聚体形式（由一个66 kDa亚基和一个51 kDa亚基组成）来估计每个突变蛋白的稳定性。所得的表型数据提供了一种“遗传”方法，用于鉴定对蛋白质功能或稳定性重要的氨基酸侧链，以及位于蛋白质表面的侧链。使用几种HIV-1 RT晶体结构来评估突变分析。我们的遗传图谱与晶体结构相关性良好。将我们的表型数据与晶体学数据相结合，使我们能够研究遗传定义的关键残基。重要的功能残基位于酶活性位点附近。许多对RT稳定性重要的残基参与蛋白质内部潜在的氢键或疏水相互作用。除了能更好地理解HIV-1 RT外，这项工作还证明了饱和诱变在总体上研究蛋白质的功能、结构和稳定性方面的实用性。该策略对于研究没有晶体学数据的蛋白质应该是有用的。
