STRUCTURAL MODULARITY & PROTEIN FUNCTION IN CYTOCHROME C

这项工作的长期目标是理解 对蛋白质结构和功能很重要的相互作用。 蛋白质折叠，获得最终的、三维的三级结构 从初级氨基酸序列得到的结构，是 将编码在DNA中的遗传信息转化为功能 产品。这种三级结构是非常特殊的：一个 单一蛋白质中的单一氨基酸残基可引起转化或 消除原本健康的细胞的生存能力。 许多不同的技术正在被用来研究这一点 有问题。定点突变已被用来确定 特定残基对特定的折叠或功能的重要性 蛋白。定向随机突变，定点定向的一种形式 突变，是一种将一个或多个残基突变的技术 在一次实验中转化成所有可能的氨基酸，这使得 有关残留物的“可替代性”的更多信息，请访问 就一次。在另一种形式的突变中，寡核苷酸导向 诱变，完整的蛋白质片段-二级结构， 超二级结构或属性域-使用单个 寡核苷酸。层次结构或模块化的程度 从这种类型的实验中可以确定蛋白质中是否存在蛋白质。 这项提案中描述的研究将应用这些 技术导向、随机诱变和寡核苷酸导向 致突变-异L-细胞色素c，一个重要的高度保守的 酿酒酵母中的电子传递蛋白 以确定对结构的影响或突变 这种蛋白质的功能。寡核苷酸导向的突变是 已经在这个系统中使用了，但随机突变技术 必须首先开发。因此，这项工作有四个具体目标：1) 继续进行寡核苷酸引导的环和螺旋突变 ISO-L-细胞色素c确定哪些区域结构上或 功能上的重要性；2)研究光谱和动力学 在体内和体外确定突变蛋白的性质 每一种突变都会影响蛋白质的结构和功能；3)发展出一种 在异型L细胞色素c中进行定向、随机突变的系统； 4)利用随机诱变进一步探索结构和功能 这种蛋白质中的关系。 定向随机突变的结果和 寡核苷酸导向的突变以前并不是始终如一的 在此系统中应用，但将导致更好地理解 参与折叠的特定残基或残基基团以及 细胞色素c的功能；此外，推导出的任何一般折叠规则 细胞色素c可能适用于其他蛋白质的折叠。

The long-term objective of this work is to understand the interactions which are important for protein structure and function. Protein folding, the acquisition of a final, three-dimensional tertiary structure from a primary amino acid sequence, is the final step in translating the genetic message encoded in the DNA into a functional product. This tertiary structure is very specific: the mutation of a single amino acid residue in a single protein can cause transformation or eliminate viability of an otherwise healthy cell. Many different technologies are being used to study this problem. Site-directed mutagenesis has been used to determine the importance of specific residues for the folding or function of a specific protein. Directed, random mutagenesis, one form of site-directed mutagenesis, is a technique in which a residue or residues are mutated into all possible amino acids In a single experiment, which allows much more Information about a residue's "replaceability" to be determined at one time. In another form of mutagenesis, oligonucleotide-directed mutagenesis, whole protein segments - secondary structures, supersecondary structures, or domains - are modified with a single oligonucleotide. The degree of hierarchical structure or modularity present In a protein can be determined from this type of experiment. The research described in this proposal will apply these techniques - directed, random mutagenesis and oligonucleotide-directed mutagenesis - to iso-l-cytochrome c, an important and highly conserved electron transport protein in the yeast Saccharomyces cerevisiae, In order to determine the affect or the mutations on the structure and function of this protein. Oligonucleotide-directed mutagenesis is already in use in this system, but the techniques of random mutagenesis must be first developed. This work, then, has four specific goals: 1) To continue oligonucleotide-directed mutagenesis of loops and helices In Iso-l-cytochrome c to determine which regions are structurally or functionally important; 2) To study the spectroscopic and kinetic properties or the mutant proteins in vivo and in vitro to determine how each mutation affects the protein structure and function; 3) To develop a system for performing directed, random mutagenesis in Iso-l-cytochrome c; and 4) To use random mutagenesis to further probe structure and function relationships in this protein. The results of directed, random mutagenesis and oligonucleotide-directed mutagenesis have not before been consistently applied in this system, but will lead to a better understanding of the specific residues or groups of residues involved in the folding and function of cytochrome c; furthermore, any general folding rules deduced for cytochrome c may be applicable to the folding of other proteins.