SUBUNIT INTERACTIONS IN DNA BINDING PROTEINS

与核酸结合的蛋白质参与了许多重要的 生物过程，包括复制，转录， 翻译和重组事件。 它们对核酸的亲和力 酸通常取决于某些代谢物的水平，具体 离子和盐浓度。 虽然许多人的约束力 DNA结合蛋白质与其特异性识别序列的结合， 这些蛋白质的聚集状态已得到充分表征 还没有被系统调查过 拟议 研究重点是三种模型蛋白质系统，它们可以识别 双链脱氧核糖核酸的特定序列。 这些蛋白质中有两种是阻遏蛋白质，lac和trp 阻遏物 两者都是低聚物（分别为四聚体和二聚体） 在微摩尔浓度。 第三个制度是限制 内切核酸酶EcoRI，其可以形成四聚体和二聚体。 通过测定这些分子的亚基缔合自由能， 不同离子和代谢物条件下的寡聚体亚基 浓度，我们将确定是否以及如何相互作用， 亚基界面参与调节蛋白质的亲和力 找DNA 这些研究中采用的平衡方法将 是那些高流体静压和温度变化的 耦合高灵敏度多频稳态 荧光测定法 高的静水压力会破坏 许多蛋白质亚基相互作用。 因此，高压 荧光研究这些低聚物标记的长寿命， 时间荧光探针将允许测定 通过效应分子（辅阻遏物、抗- 诱导剂和诱导剂）、DNA结合和离子强度， 亚基缔合平衡条件下使用纳摩尔 蛋白质浓度。 此外，研究的内在 色氨酸荧光偏振和寿命，作为函数 温度、压力和结扎状态将产生信息 蛋白质构象和动力学的变化 与效应分子和DNA的结合有关 序列的 这些动力学和热力学数据将为我们的 了解转移的物理机制， 将信息从一个亚基结合到其他亚基， 生物学上重要的寡聚蛋白。

Proteins that bind to nucleic acids are involved in many important biological processes, including replication, transcription, translation, and recombination events. Their affinity for nucleic acids often depends upon levels of certain metabolites, specific ions, and salt concentration. While the binding of a number of DNA binding proteins to their specific recognition sequences has been well characterized, the aggregation state of these proteins has not been the object of systematic investigation. The proposed research focuses on three model protein systems which recognize specific sequences of double stranded deoxyribonucleic acids. Two of these proteins are repressor proteins, the lac and trp repressors. Both are oligomers (tetramer and dimer, respectively) at micromolar concentrations. The third system is the restriction endonuclease, EcoRI, which can form both tetramers and dimers. By determining the free energy of subunit association for these oligomer subunits under varying conditions of ion and metabolite concentration, we will determine if and how interactions at the subunit interface are involved in regulation the proteins' affinity for DNA. The equilibrium methods employed in these studies will be those of high hydrostatic pressure and temperature variations coupled to high sensitivity multi-frequency and steady state fluorometry. High hydrostatic pressure is known to destabilize many protein subunit interactions. Thus, high pressure fluorescence studies of these oligomers labelled with long-life- time fluorescent probes will allow for the determination of the effects of ligation by effector molecules (corepressors, anti- inducers, and inducers), DNA binding, and ionic strength upon subunit associations under equilibrium conditions using nanomolar protein concentrations. Additionally, studies of the intrinsic tryptophan fluorescence polarization and lifetime, as a function of temperature, pressure, and ligation state will yield information as to the changes in protein conformation and dynamics associated with the binding of effector molecules and DNA sequences. This dynamic and thermodynamic data will add to our understanding of the physical mechanisms of the transfer of binding information from one subunit to the others in these biologically important oligomeric proteins.