SOLUTION STRUCTURE AND FUNCTION OF MUTANT CRO REPRESSORS

Lambda cro阻遏突变体的功能结构将是 使用技术在原子分辨率级别的溶液中进行分析 二维核磁共振波谱和距离几何学。生产过剩的人 一种经过基因工程改造的质粒菌，因此在 抑制子可以用任何将被用来产生 突变抑制物用于研究战略突变对细胞生长的影响 抑制物的结构、稳定性和特异性。解决方案 突变抑制物的结构将在大约1埃时确定。 基于时变NOESY距离约束的分辨 实验，结合距离几何计算，生成 三维结构。抑制者：运算符复合体也将 由二维核磁共振研究，试图定义原子间的精确接触 DNA表面：蛋白质界面。 基于当前的特定DNA识别模型，互补 操作员DNA序列中的突变将被合成，以尝试 恢复突变抑制子的亲和力和特异性。这将导致 为设计和工程能够结合的阻遏蛋白 任何预先确定的DNA序列。这个项目的长期目标是 在原子水平上对序列特定DNA的详细理解 抑制蛋白的识别，以及基本的理解 氨基酸序列对蛋白质结构和稳定性的影响。

The functional architecture of Lambda cro repressor mutants will be analyzed in solution at the level of atomic resolution using the techniques of two-dimensional NMR spectroscopy and distance geometry. An overproducer plasmid strain that has been genetically engineered so that any position in the repressor can be replaced with any amino acid will be used to produce mutant repressors for studying the effects of strategic mutations on the structure, stability, and specificity of the repressor. The solution structure of the mutant repressors will be determined at ca. 1 Angstrom resolution using the distance constraints of time-dependent NOESY experiments, combined with distance geometry calculations, to generate the three-dimensional structure. Repressor:operator complexes will also be studied by 2DNMR in an attempt to define the precise atomic contacts at the surface of the DNA:protein interface. Based on current models of specific DNA recognition, complementary mutations in the operator DNA sequence will be synthesized in an attempt to restore the affinity and specificity of mutant repressors. This will lead to the design and engineering of repressor proteins capable of binding to any pre-determined DNA sequences. The long term goal of this project is a detailed understanding, at the atomic level, of sequence-specific DNA recognition by repressor proteins, as well as a fundamental understanding of the effects of amino acid sequence on protein structure and stability.