GENETIC REVERSION AND NMR AS PROBES OF PROTEIN STRUCTURE

许多蛋白质的序列和结构都是已知的，但它们之间的关系 这些性质之间的关系还没有被很好地理解。一种常见的蛋白质 结构基序是α-螺旋之间的相互作用。澄清 管理这种相互作用的规则是实现蛋白质目标的先决条件 工程学--蛋白质结构和功能的从头设计。什么时候 蛋白质设计是可能的，新的、复杂的药物可以用来合成 生物常见的化学，但合成有机却有问题 化学反应。设计还将允许改进治疗性蛋白质 例如TPA。 我的研究计划是寻找管理阿尔法螺旋相互作用的规则 在蛋白质细胞色素c中，使用随机、基因内、 第二位点逆转(RISR)突变改变螺旋，和强大的 一种新技术，二维核磁共振(2-DNMR)，用于 确定改变后的蛋白质的溶液结构。 与定点突变不同的是，随机突变几乎不做任何假设 关于单个残基的作用，并允许系统告诉 研究人员发现序列中哪些元素对结构很重要。 然而，大多数随机突变是沉默的。RISR突变，这是一种 两个阶段的过程，消除了沉默的随机突变。第一，随机性 产生的突变会扰乱基因功能。然后是第二套 随机突变被引入到不同的位置，它复活了 功能。 拟议研究的具体目标是通过以下方式产生RISR突变 分开插入横跨4个的随机寡核苷酸盒 连续密码子进入细胞色素c的N端和C端的α-螺旋。 用2-D测定的第二位反转剂的溶液结构 核磁共振，将导致关于螺旋序列要求的建议 互动。然后，这些建议将通过制作站点定向的 变种人。酵母细胞色素c系统，我已经用了5年了 具有产生RISR突变体所需的特征。

Sequences and structures for many proteins are known, but the relationship between these properties is not well understood. A common protein structural motif is the interaction between alpha-helices. Elucidating rules governing such interactions is a prerequisite to the goal of protein engineering--de novo design of protein structure and function. When protein design is possible, new, complex drugs may be synthesized using chemistry common to biology, but problematic to synthetic organic chemistry. Design would also allow improvement of therapeutic proteins such as TPA. My research plan is to search for rules governing alpha-helical interaction in the protein, cytochrome c, using the technique of random, intragenic, second-site reversion (RISR) mutagenesis to alter helices, and the powerful new technique, 2-dimensional nuclear magnetic resonance (2-D NMR), to determine the solution structure of the altered proteins. Unlike site-directed mutagenesis, random mutagenesis makes few assumptions about the role of single residues and allows the system to tell the investigator which elements of sequence are important to structure. However, most random mutations are silent. RISR mutagenesis, which is a two stage process, eliminates silent, random mutations. First, random mutations are produced which disrupt gene function. Then a second set of random mutations is introduced at a different site, which resurrect function. The specific aim of the proposed research is to generate RISR mutations by separate insertion of random oligonucleotide cassettes spanning 4 contiguous codons into the N- and C-terminal alpha-helices of cytochrome c. The solution structure of second-site revertants, as determined using 2-D NMR, will lead to proposals concerning sequence requirements for helix interactions. These proposals will then be tested by making site-directed mutants. The yeast cytochrome c system, which I have worked with for 5 years, possesses the features necessary to produce RISR mutants.