He J J, Matthews K S
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251.
J Biol Chem. 1990 Jan 15;265(2):731-7.
Mutations in the tryptophan-binding site of the trp repressor have been generated using site-directed mutagenesis. The selection of sites for alteration was based on the three-dimensional x-ray crystallographic structure (Schevitz, R. W., Otwinowski, Z., Joachimiak, A., Lawson, C. L., and Sigler, P. B. (1985) Nature 317, 782-786). The changes generated include Thr-44 to Ala (T44A), Arg-54 to Leu (R54L), Arg-54 to Lys (R54K), Arg-84 to Leu (R84L), and Arg-84 to Lys (R84K). The mutant proteins were purified and characterized in detail for their binding properties. Both tryptophan and operator DNA affinities for all five mutants were decreased. The R84L, R54K, and R54L mutants exhibited increases in Kd for operator DNA relative to wild-type repressor ranging from approximately 10(3) to approximately 10(4), while R84K and T44A exhibited increases of 10- to 100-fold. This diminution in DNA binding activity derives at least in part from diminished affinity for tryptophan, although decreased affinity for nonspecific DNA was also observed for these mutant proteins. Tryptophan binding was not detectable by equilibrium dialysis for most of the mutant proteins, but this activity was measurable for several of the altered proteins by monitoring the fluorescence decrease associated with the displacement of 1-anilino-8-naphthalenesulfonate from the tryptophan-binding site (Chou, W.-Y., and Matthews, K. S. (1989) J. Biol. Chem. 264, 18314-18319). These measurements revealed that tryptophan bound to R84K, T44A, and R84L repressors with Kd values 1.5- to 13-fold higher than that for wild-type repressor. It was not possible to detect tryptophan binding to R54K and R54L even using the fluorescence assay. Circular dichroism spectra demonstrated that the mutants and the wild-type repressor possess similar secondary structural features. The results of this selected substitution in the tryptophan-binding site are readily interpreted based on the x-ray structural analysis.
利用定点诱变技术在色氨酸阻遏物的色氨酸结合位点产生了突变。改变位点的选择基于三维X射线晶体结构（谢维茨，R.W.，奥特维诺夫斯基，Z.，乔阿奇米亚克，A.，劳森，C.L.，和西格ler，P.B.（1985年）《自然》317，782 - 786）。产生的变化包括苏氨酸-44突变为丙氨酸（T44A）、精氨酸-54突变为亮氨酸（R54L）、精氨酸-54突变为赖氨酸（R54K）、精氨酸-84突变为亮氨酸（R84L）以及精氨酸-84突变为赖氨酸（R84K）。对突变蛋白进行了纯化，并详细表征了它们的结合特性。所有五个突变体对色氨酸和操纵基因DNA的亲和力均降低。相对于野生型阻遏物，R84L、R54K和R54L突变体对操纵基因DNA的解离常数（Kd）增加了约10³至约10⁴倍，而R84K和T44A增加了10至100倍。DNA结合活性的这种降低至少部分源于对色氨酸亲和力的降低，尽管这些突变蛋白对非特异性DNA的亲和力也有所下降。对于大多数突变蛋白，通过平衡透析无法检测到色氨酸结合，但通过监测与1 - 苯胺基 - 8 - 萘磺酸盐从色氨酸结合位点的置换相关的荧光降低，可测量几种改变蛋白的这种活性（周，W.-Y.，和马修斯，K.S.（1989年）《生物化学杂志》264，18314 - 18319）。这些测量结果表明，色氨酸与R84K、T44A和R84L阻遏物结合的Kd值比野生型阻遏物高1.5至13倍。即使使用荧光测定法，也无法检测到色氨酸与R54K和R54L的结合。圆二色光谱表明，突变体和野生型阻遏物具有相似的二级结构特征。基于X射线结构分析，很容易解释在色氨酸结合位点进行这种选定取代的结果。
