CALORIMETRIC STUDIES OF MAT A2 HOMEODOMAIN

同源结构域是一类DNA结合蛋白结构域，具有 在转录和发育控制中的重要作用 真核生物，在某些情况下参与了人类的肿瘤发生。我们 正在使用酵母的MAT a2同源结构域(Li等人，1995，Science 270,262-269)作为模型系统来表征 蛋白质折叠及其与序列特异性DNA的结合 Motif。使用差示扫描和等温滴定 量热法，我们测量了热焓、熵、热容和 野生型过程的Gibbs自由能变化 A2同源结构域序列(Carra&Privalov，1997，生物化学36， 526-)。蛋白质-DNA的相互作用是由热能驱动的 生理温度。蛋白质折叠和DNA结合是 连接的，如其他几种DNA结合蛋白。一种比较 游离蛋白质和DNA结合蛋白质的圆二色谱 物种揭示蛋白质结构的形成是由DNA诱导的 有约束力的。因此，为关联而测量的能量包括 组件由于折叠。我们目前正将这些研究扩展到 A2同源结构域的突变版本。定点突变是 用于创建含有丙氨酸的同源结构域的版本 DNA识别螺旋中的替换，移除了重要的 从联系人到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 替换。蛋白质的N末端臂的缺失，这是 包裹在复合体中DNA的背面，导致了剧烈的 DNA结合亲和力丧失。MATA2同源结构域也可以与DNA结合 与MATA1同源结构域协同，作为异源二聚体。这 蛋白质之间的相互作用，以及异源二聚体与DNA的结合，将 被研究使用孤立的 同源域和基因产生的两者的融合。

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.