STRUCTURE AND FUNCTION OF VARIANT LAMBDA CRO PROTEINS

蛋白质结构和DNA识别的分子决定因素将是 研究了lambda Cro蛋白的工程变体。二聚体 Cro的界面，由来自每个亚基的一条链组成的Beta带， 对折叠、稳定性和DNA识别至关重要。工程学和 诱变工作将集中在该地区，以确定和分离 特定残基和结构元素在每一个 以上功能。Cro的一个新的单体变体家族已经被 其中野生型二聚体界面已被替换为 一个精心设计的贝塔发夹转弯。最近2D核磁共振实验的数据是 与贝塔发夹的存在相吻合。克鲁斯的水晶 得到了衍射率大于1.6埃的单体。这个 这些蛋白质的结构、稳定性和DNA结合活性将 与引入的氨基酸残基序列相关，形成 转弯。局部结构单元的结构与柔度 将根据它们在保持高效率方面的作用来量化 两个简单单体蛋白中相互作用基团的浓度 二聚体DNA结合蛋白与其结合蛋白的结构及复合体 对称DNA位点。 合理设计、随机诱变和遗传选择的结合 将被用来构建符合特定功能标准的新蛋白质。 这些蛋白质的特征将被用来解决特定的问题 蛋白质结构和DNA识别。不同的组合如何 氨基酸残基影响贝塔带的硬度和延伸性 贝塔发夹的特性是什么？什么是耦合自由能 当结合到时归因于相同亚基之间的特定连锁 一个二元对称的操作入口站点？可以使用不同的链接来改变 特异性不是通过改变与DNA直接接触的残基，而是通过改变与DNA直接接触的残基来实现的 通过修改悬挂它们的框架？回答 像这样的问题将加深我们对蛋白质结构和 功能。

The molecular determinants of protein structure and DNA recognition will be studied with engineered variants of the lambda Cro protein. The dimer interface of Cro, a beta ribbon consisting of a strand from each subunit, is crucial for folding, stability, and DNA recognition. Engineering and mutagenesis efforts will be focused in this region to identify and isolate the roles of particular residues and structural elements in each of the above functions. A family of a novel monomeric variants of Cro has been constructed in which the wild type dimer interface has been replaced with a designed beta-hairpin turn. Data from recent 2D NMR experiments are consistent with the presence of the beta hairpin. Crystals of a Cro monomer have been obtained which diffract to beyond 1.6 Angstroms. The structures, stabilities, and DNA binding activities of these proteins will be correlated with the sequences of amino acid residues introduced to form the turns. The structure and flexibility of elements of local structure will be quantified in terms of their roles in maintaining high effective concentrations of interacting groups in both a simple monomeric protein structure and a complex between a dimeric DNA binding protein and its symmetric DNA site. A combination of rational design, random mutagenesis and genetic selection will be used to build new proteins which meet specific functional criteria. These proteins will be characterized and used to address specific questions of protein structure and DNA recognition. How do different combinations of amino acid residues affect the stiffness and extension of a beta ribbon or the properties of a beta hairpin? What coupling free energy can be attributed to a particular linkage between identical subunits when bound to a dyad symmetric operator site? Can variant linkages be used to alter specificities not by changing the residues in direct contact with DNA but by modifying the framework from which they are suspended? Answers to questions like these will refine our understanding of protein structure and function.