STRUCTURE/FUNCTION OF VARIANT LAMBDA CRO PROTEINS

蛋白质结构和DNA识别的分子决定因素将是 用λ Cro蛋白的工程变体进行研究。 二聚体 Cro的界面，由来自每个亚基的链组成的β带， 对于折叠、稳定性和DNA识别至关重要。 工程和 诱变的努力将集中在这一地区，以确定和分离 在每一个特定的残基和结构元件的作用， 以上功能。 一个新的Cro单体变体家族已经被发现， 构建其中野生型二聚体界面已被替换为 一个设计好的β发夹弯 来自最近的2D NMR实验的数据是 与β发夹的存在一致 十字架的结晶 已经获得了聚合度超过1.6埃的单体。 的 这些蛋白质的结构、稳定性和DNA结合活性将 与引入以形成的氨基酸残基的序列相关 转弯 局部结构元素的结构与柔性 将根据其在维持高效率方面的作用进行量化 在一个简单的单体蛋白质中， 结构和二聚体DNA结合蛋白与其 对称DNA位点。 合理设计、随机诱变和遗传选择相结合 将用于构建满足特定功能标准的新蛋白质。 这些蛋白质将被表征并用于解决特定的问题 蛋白质结构和DNA识别。 不同组合的 氨基酸残基影响β带的硬度和延伸，或 β发夹的特性？ 耦合自由能是多少 这归因于相同亚基之间的特定连接， 一个二分体对称算子位点 变异的连接是否可以用来改变 特异性不是通过改变与DNA直接接触的残基， 通过修改他们被悬挂的框架？ 的答案 像这样的问题将完善我们对蛋白质结构的理解， 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.