STUDIES ON THE CYCLIC AMP RECEPTOR PROTEIN OF E COLI

环腺苷酸受体蛋白（CRP）作为一种调节因子， 在E.杆菌 cAMP结合结果 CRP所需的构象变化。 MBRS项目涉及 CRP Gly-200突变为Phe。 这种甘氨酸提供了所需的转向 为了使相邻的β-11和β-12形成合适的构象， 股。 突变将GGTAAA（Gly-Lys）转化为TTTAAA（Phe-Lys） 提供大体积的氨基酸残基以干扰构象 参与了一种重要的β折叠结构， CRP的C末端结构域。 这两个基地的变化也形成了一个德拉尔 限制网站，以监测成功的建设， 在小型印刷机中进行所需的突变。 这是有用的，因为不清楚 该突变将导致CRP活性的变化， 通过平板测定进行表型选择。 项目合理 直接向前，并将提供MBRS学者与一系列的， 在现代分子生物学中具有重要意义的实验经验。 这些 包括噬菌粒生产和分离所需的程序， 单链DNA、定点突变和双脱氧DNA 测序、转化、CRP突变体的选择、β 半乳糖苷酶活性，将突变体CRP纯化至均一 以及几种用于表征 CRP的结构/功能特性。 学生将开始他/她的任期在实验室的协助和 向一个研究生学习技术 在某个时候 在他们的学徒期间（当我评估他们的进步时），他们将开始 更独立于他们的直接导师工作。 我将讨论的 结果，提出建议，协助解释数据，批评 在适当的时候。 学生将沿着参加实验讨论 与我的其他学生，并可能参加ASBCMB会议，并预计 在MBRS全国会议上介绍这项研究。

The cyclic AMP receptor protein (CRP) acts as a regulatory factor for the transcription of more than 20 genes in E. coli. Binding of cAMP results in a required conformational change in CRP. The MBRS project involves mutation of CRP Gly-200 to Phe. This glycine provides the turn required for the appropriate conformation of the adjacent Beta-11 and Beta-12 strands. The mutation will convert GGTAAA (Gly-Lys) to TTTAAA (Phe-Lys) providing a bulky amino acid residue to interfere with the conformation involved in what is apparently an essential Beta sheet structure in the C-terminal domain of CRP. The two bases change also forms a Dral restriction site for monitoring the successful construction of the desired mutation in minipress. This is useful since it is not clear that the mutation will cause a change in CRP activity which would allow for phenotypic selection by a plate assay. The project is reasonable straight forward and will provide the MBRS scholar with a series of experimental experiences important in current molecular biology. These include the procedures required for phagemid production and isolation of the single stranded DNA, site specific mutagenesis and dideoxy DNA sequencing, transformation, selection of CRP mutants, assays for Beta galactosidase activity, purification of the mutant CRPs to homogeneity and the several in vitro assays used to characterize the structure/function properties of CRP. The student will begin his/her tenure in the laboratory by assisting and learning techniques from one of the graduate students. At some time during their apprenticeship (as I assess their progress) they will begin to work more independently of their immediate mentor. I discuss the results, make suggestions, assist in interpreting the data, criticize where appropriate. The student will participate in lab discussions along with my other students and may attend an ASBCMB Meeting and are expected present the research at an MBRS national meeting.