Hickman A B, Palmer I, Engelman A, Craigie R, Wingfield P
Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1994 Nov 18;269(46):29279-87.
A deletion derivative of the integrase protein from human immunodeficiency virus type-1 (HIV-1) consisting of the central core domain (amino acids 50-212) has been characterized biophysically and biochemically. This deletion mutant is of particular interest for structural studies as it can carry out the disintegration reaction suggesting the presence of an active site and, under certain conditions, is more soluble than full-length integrase. The circular dichroism and fluorescence of the deletion mutant and the 288-residue full-length integrase were similar, indicating that the core residues maintain similar overall conformations in both proteins. The deletion mutant is approximately 10% more alpha-helical than the full-length protein. Analytical centrifugation demonstrated that both proteins undergo monomer-dimer association although the truncated protein showed slightly less tendency to dimerize; the dissociation constants were 2.5 x 10(-5) M for the full-length protein and 8.0 x 10(-5) M for the truncated protein. The disintegration activity of both proteins was also compared. Although a higher concentration of the truncation mutant was required for optimal activity, the mutant did not have altered pH or Mn2+ requirements relative to the full-length protein. The combined biophysical and enzymatic studies suggest that this truncated form of HIV-1 integrase is likely to be useful for structural studies.
一种来自人类免疫缺陷病毒1型（HIV-1）的整合酶蛋白缺失衍生物，由中央核心结构域（氨基酸50 - 212）组成，已通过生物物理和生化方法进行了表征。这种缺失突变体对于结构研究特别有意义，因为它能够进行解体反应，表明存在活性位点，并且在某些条件下比全长整合酶更易溶解。缺失突变体和288个残基的全长整合酶的圆二色性和荧光相似，表明核心残基在两种蛋白质中保持相似的整体构象。缺失突变体的α-螺旋含量比全长蛋白质大约高10%。分析超速离心表明，两种蛋白质都经历单体 - 二聚体缔合，尽管截短的蛋白质显示出稍低的二聚化倾向；全长蛋白质的解离常数为2.5×10⁻⁵ M，截短蛋白质的解离常数为8.0×10⁻⁵ M。还比较了两种蛋白质的解体活性。尽管截短突变体达到最佳活性需要更高的浓度，但相对于全长蛋白质，该突变体对pH或Mn²⁺的需求没有改变。综合的生物物理和酶学研究表明，这种HIV-1整合酶的截短形式可能对结构研究有用。
