GENETIC REVERSION AND NMR AS PROBES OF PROTEIN STRUCTURE

许多蛋白质的序列和结构是已知的，但它们之间的关系是未知的。 这些属性之间的关系还没有很好的理解。 共同的蛋白质 结构基序是α-螺旋之间的相互作用。 阐明 控制这种相互作用的规则是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。 当 蛋白质设计是可能的，新的，复杂的药物可以合成使用 生物学中常见的化学，但合成有机化学中存在问题 化学. 设计还可以改善治疗性蛋白质 例如TPA。 我的研究计划是寻找α螺旋相互作用的规律 在蛋白质中，细胞色素c，使用随机的，基因内的， 第二位点回复突变（RISR）来改变螺旋， 二维核磁共振（2-D NMR）新技术， 确定改变的蛋白质的溶液结构。 与定点突变不同，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 研究序列中哪些元素对结构重要。 然而，大多数随机突变是沉默的。 RISR诱变，这是一种 两阶段过程，消除沉默，随机突变。 第一，随机 产生了破坏基因功能的突变。 然后是第二组 在不同的位点引入随机突变， 功能 拟议研究的具体目的是通过以下方式产生RISR突变： 跨越4个核苷酸的随机寡核苷酸盒的单独插入 将连续密码子插入细胞色素c的N-和C-末端α-螺旋中。 第二位点回复突变体的溶液结构，如使用2-D 核磁共振，将导致有关螺旋序列要求的建议 交互. 然后，这些建议将通过进行现场指导来进行测试。 变种人 酵母细胞色素c系统，我已经与5 年，具有产生RISR突变体所必需的特征。

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.