GENETIC REVERSION AND NMR AS PROBES OF PROTEIN STRUCTURE

基因逆转和核磁共振作为蛋白质结构的探针

• 批准号: 3467693
• 负责人: GARY JOSEPH PIELAK
• 金额: $ 9.8万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3467693
• 关键词: Escherichia coli; Saccharomyces cerevisiae; conformation; cytochrome c; disulfide bond; nuclear magnetic resonance spectroscopy; nucleic acid probes; oligonucleotides; plasmids; protein engineering; protein structure; protein structure function; site directed mutagenesis; transposon /insertion element

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.

许多蛋白质的序列和结构是已知的，但是这种关系 在这些属性之间尚不清楚。 一种常见的蛋白质 结构基序是α-螺旋之间的相互作用。 阐明 控制这种互动的规则是蛋白质目标的先决条件 工程 - 蛋白质结构和功能的新发现。 什么时候 蛋白质设计是可能的，可以使用新的复杂药物使用 生物学常见的化学，但对合成有机物有问题 化学。 设计还可以改善治疗蛋白 例如TPA。 我的研究计划是搜索管理α螺旋互动的规则 在蛋白质中，细胞色素c使用随机，内部的技术 第二站点恢复（RISR）诱变以改变螺旋和功能 新技术，二维核磁共振（2-D NMR）到 确定改变的蛋白质的溶液结构。 与位置定向的诱变不同，随机诱变很少有假设 关于单个残留物的作用，并允许系统告诉 研究者哪些序列要素对结构很重要。 但是，大多数随机突变是沉默的。 RISR诱变，这是 两个阶段过程，消除了无声的随机突变。 首先，随机 产生破坏基因功能的突变。 然后第二组 随机突变是在不同的地点引入的，该突变是复活的 功能。 拟议研究的具体目的是通过 跨越4的随机寡核苷酸盒的单独插入4 连续的密码子进入细胞色素c的N-和C末端α-螺旋。 使用2-D确定的第二位恢复物的溶液结构 NMR，将导致有关螺旋序列要求的建议 互动。 然后将通过定向地进行测试这些建议 突变体。 我曾与之合作的5个酵母细胞色素C系统 几年，具有产生RISR突变体所需的特征。