CALORIMETRIC STUDIES OF MAT A2 HOMEODOMAIN

MAT A2 同源域的量热研究

• 批准号: 6122033
• 负责人: JOHN H CARRA
• 金额: --
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-05 至 1998-08-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6122033
• 关键词: biological products; biomaterials; biomedical resource; education; human tissue; model design /development; nucleic acids; proteins; technology /technique development

Homeodomains are a class of DNA-binding protein domains which have important roles in control of transcription and development in eukaryotes, and in some cases are involved in human oncogenesis. We are using the MAT a2 homeodomain of yeast (Li et al., 1995, Science 270, 262-269) as a model system to characterize the thermodynamics of protein folding and sequence-specific DNA binding by this protein motif. Using differential scanning and isothermal titration calorimetry, we measured the enthalpy, entropy, heat capacity, and Gibbs free energy changes of these processes for the wild-type sequence of a2 homeodomain (Carra & Privalov, 1997,Biochemistry 36, 526-535). The protein-DNA interaction is enthalpically driven at physiological temperatures. Protein folding and DNA binding are linked, as for several other DNA binding proteins. A comparison of the circular dichroism spectra of the free and DNA-bound protein species revealed that formation of protein structure is induced by DNA binding. The energies measured for association therefore include a component due to folding. We are currently extending these studies to mutant versions of the a2 homeodomain. Site-directed mutagenesis was used to create versions of the homeodomain that contain alanine substitutions in the DNA recognition helix, removing important contacts to DNA. Changes in the core packing of the protein were also made to assay the importance of certain conserved residues to folding, and the ability of the protein core to accept compensating substitutions. Deletion of the N-terminal arm of the protein, which wraps around the back of the DNA in the complex, results in a drastic loss of DNA binding affinity. The MATa2 homeodomain can also bind DNA cooperatively with the MATa1 homeodomain, as a heterodimer. This protein-protein interaction, and DNA binding by the heterodimer, will be studied using the isolated homeodomains and a genetically produced fusion of the two.

同源结构域是一类DNA结合蛋白结构域， 在控制转录和发育中的重要作用， 真核生物，并且在某些情况下参与人类肿瘤发生。 我们 正在使用酵母的MAT α 2同源结构域（Li等人，1995年，科学 270，262-269）作为模型系统来表征 蛋白质折叠和该蛋白质的序列特异性DNA结合 母题 采用差示扫描和等温滴定法 量热法，我们测量了焓，熵，热容， 这些过程的吉布斯自由能变化为野生型 α 2同源结构域的序列（Carra和Privalov，1997，Biochemistry 36， 526-535）。 蛋白质与DNA的相互作用是由遗传驱动的， 生理温度。 蛋白质折叠和DNA结合是 连接，就像其他几种DNA结合蛋白一样。 的比较 游离蛋白质和DNA结合蛋白质的圆二色光谱 物种揭示了蛋白质结构的形成是由DNA诱导的 约束力 因此，为缔合测量的能量包括 由于折叠。 我们目前正在将这些研究扩展到 A2同源结构域的突变形式。 定点诱变 用来制造含有丙氨酸的同源结构域 DNA识别螺旋中的取代，去除重要的 接触DNA。 蛋白质核心包装的变化也是 用来分析某些保守残基对折叠的重要性， 以及蛋白质核心接受补偿的能力 替代品 蛋白质的N-末端臂的缺失， 缠绕在复合体中DNA的后部，导致剧烈的 丧失DNA结合亲和力。 MATa 2同源结构域也可以结合DNA 与MATa 1同源结构域协同作用，作为异二聚体。 这 蛋白质-蛋白质相互作用和异二聚体与DNA的结合， 使用隔离的 同源结构域和基因产生的两者的融合。