Benevides J M, Weiss M A, Thomas G J
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City 64110.
J Biol Chem. 1994 Apr 8;269(14):10869-78.
The lambda repressor exhibits structural characteristics of lock and key complementary through the helix-turn-helix motif, and of induced fit by virtue of DNA-dependent folding of the N-terminal arm. In both cases, molecular recognition is mediated by direct contacts between amino acids and DNA bases. The extent to which such contacts function as discrete elements in a protein-DNA recognition code is not known. Because of the relevance of protein recognition to the broader issue of protein design, and because the lambda system serves as a prototype for gene regulation, we have employed laser Raman and 1H NMR spectroscopy to compare free and operator-bound structures of lambda repressor variants which are known to exhibit altered DNA-binding specificities. Experimental design is based upon a previous biochemical study of mutations in the repressor N-terminal arm (K4Q) and helix-turn-helix motif (G48S) (Nelson, H. C. M., and Sauer, R. T. (1986) J. Mol. Biol. 192, 27-38). These mutations, which were originally isolated by loss of function (K4Q) and second-site reversion (G48S), are of particular interest in light of their complex effects on sequence specificity at multiple positions in the operator site (Benson, N., Adams, C., and Youderian, P. (1992) Genetics 130, 17-26). Laser Raman and 1H NMR spectra of repressor variants carrying one (G48S) or two mutations (K4Q/G48S) are similar to those of the native wild type repressor and are in accord with the x-ray crystal structure. Remarkably, however, the complexes of wild type and mutant repressors exhibit extensive differences both in the global DNA structure and in the environments of key functional groups along the major groove. By demonstrating that single amino acid substitutions can induce global reorganization of a protein-DNA interface, the present results establish that repressor-operator recognition in solution cannot be explained in terms of a simple recognition code.
λ阻遏蛋白通过螺旋-转角-螺旋基序展现出锁钥互补的结构特征，并借助N端臂的DNA依赖性折叠呈现出诱导契合。在这两种情况下，分子识别都是由氨基酸与DNA碱基之间的直接接触介导的。这种接触在蛋白质-DNA识别密码中作为离散元件发挥作用的程度尚不清楚。由于蛋白质识别与蛋白质设计这一更广泛问题的相关性，且λ系统作为基因调控的原型，我们采用激光拉曼光谱和¹H NMR光谱来比较已知具有改变的DNA结合特异性的λ阻遏蛋白变体的游离结构和与操纵基因结合的结构。实验设计基于先前对阻遏蛋白N端臂（K4Q）和螺旋-转角-螺旋基序（G48S）中突变进行的生化研究（纳尔逊，H.C.M.，和索尔，R.T.（1986年）《分子生物学杂志》192卷，27 - 38页）。这些突变最初是通过功能丧失（K4Q）和第二位点回复（G48S）分离得到的，鉴于它们对操纵基因位点多个位置的序列特异性具有复杂影响，因而特别引人关注（本森，N.，亚当斯，C.和尤德里安，P.（1992年）《遗传学》130卷，17 - 26页）。携带一个（G48S）或两个突变（K4Q/G48S）的阻遏蛋白变体的激光拉曼光谱和¹H NMR光谱与天然野生型阻遏蛋白的光谱相似，并且与X射线晶体结构一致。然而，值得注意的是，野生型和突变型阻遏蛋白的复合物在整体DNA结构以及沿着大沟的关键功能基团环境方面都表现出广泛差异。通过证明单个氨基酸取代可诱导蛋白质-DNA界面的整体重组，目前的结果表明溶液中阻遏蛋白与操纵基因的识别无法用简单的识别密码来解释。
