SOLUTION STRUCTURE AND FUNCTION OF MUTANT CRO REPRESSORS

λ cro阻遏物突变体的功能结构将是 在原子分辨率水平下在溶液中分析， 二维核磁共振光谱学和距离几何学。 过度生产者 质粒菌株，该质粒菌株已被遗传工程化，使得质粒中的任何位置 阻遏物可以用任何氨基酸替换 突变阻遏物，用于研究战略突变对 阻遏物的结构、稳定性和特异性。 溶液 突变阻遏物的结构将在CA确定。1埃 分辨率使用时间相关NOESY的距离约束 实验，结合距离几何计算，以生成 三维结构。 抑制子：运算符复合体也将是 2DNMR研究，试图定义在原子的精确接触， DNA：蛋白质界面。 基于目前特异性DNA识别的模型，互补 操纵基因DNA序列中的突变将被合成， 恢复突变阻遏物的亲和力和特异性。 这将导致 本发明涉及能够结合至 任何预先确定的DNA序列 该项目的长期目标是 在原子水平上对序列特异性DNA的详细了解 抑制蛋白的识别，以及对 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.