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。它们对原子核的亲和力 酸度通常取决于某些代谢物的水平， 离子和盐浓度。同时绑定了多个 DNA结合蛋白与其特定的识别序列有 经过很好的表征，这些蛋白质的聚集状态 一直不是系统调查的对象。建议数 研究集中在识别三个模型蛋白质系统上 双链脱氧核糖核酸的特定序列。 其中两种是阻遏蛋白，lac和trp。 抑制者。两者都是低聚物(分别为四聚体和二聚体) 在微摩尔浓度下。第三个制度是制约 内切酶，EcoRI，可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低聚物亚单位 集中，我们将确定是否以及如何在 亚基界面参与调节蛋白质的亲和力 为了DNA。这些研究中使用的均衡方法将 是高静水压和高温度变化的 耦合到高灵敏度多频和稳态 荧光测定法。众所周知，高静水压力会破坏稳定。 许多蛋白质亚单位的相互作用。因此，高压 这些长寿命标记低聚物的荧光研究- 时间荧光探头将允许测定 效应分子(辅阻遏子、抗癌基因)的连接作用 诱导剂和诱导剂)、DNA结合和离子强度 利用纳米分子研究平衡条件下的亚基关联 蛋白质浓度。此外，对内在性的研究 作为函数的色氨酸荧光偏振和寿命 关于温度、压力和结扎状态的信息 关于蛋白质构象和动力学的变化 与效应器分子和DNA的结合有关 序列。这些动力学和热力学数据将增加我们的 对转移的物理机制的理解 从这些亚基中的一个亚基到另一个亚基的结合信息 具有重要生物学意义的寡聚蛋白。

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.