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: 3467694
• 负责人: GARY JOSEPH PIELAK
• 金额: $ 9.9万
• 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/3467694
• 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，但它们之间的关系 这些属性之间的关系还不太清楚。 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 控制这种相互作用的规则是实现蛋白质目标的先决条件 工程——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，可以使用合成新的、复杂的药物 化学对于生物学来说很常见，但对于合成有机物来说却是有问题的 化学。 设计还可以改进治疗性蛋白质 如TPA。 我的研究计划是寻找控制α-螺旋相互作用的规则 在蛋白质、细胞色素 c 中，使用随机、基因内技术， 第二位点回复（RISR）突变改变螺旋，以及强大的 新技术，二维核磁共振（2-D NMR） 确定改变的蛋白质的溶液结构。 与定点诱变不同，随机诱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 研究人员哪些序列元素对结构很重要。 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，消除沉默的随机突变。 一、随机 产生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，从而复活 功能。 拟议研究的具体目标是通过以下方式产生 RISR 突变： 单独插入随机寡核苷酸盒跨越4 连续密码子进入细胞色素 c 的 N 端和 C 端 α 螺旋。 使用二维确定的第二位点回复体的溶液结构 NMR，将产生有关螺旋序列要求的提案 互动。 然后，这些提案将通过现场定向进行测试 突变体。 酵母细胞色素 c 系统，我已经使用它 5 年了 年，拥有产生 RISR 突变体所需的特征。