NMR Studies of Protein Side-Chain Dynamics

该项目的总体目标是研究氨基酸侧链动力学对蛋白质合成的贡献。 物理化学机制，决定了热力学的识别和协会， 蛋白质/DNA相互作用和钙结合蛋白离子结合协同性的分子基础 （CaBPs）。侧链可以对蛋白质的构型熵做出重要贡献， 调节蛋白质功能的热力学。对蛋白质工作原理的基本理解 因此需要对侧链的动态性质有深入的了解。两个模型系统， 蛋白质/DNA复合物和CaBP钙结合蛋白D9 k，已被选择用于研究，这将使关键的见解， 关于侧链动力学在蛋白质/DNA结合/识别中的作用以及 离子结合。尽管有大量的结构和热力学研究， 报告的各种蛋白质/DNA系统，关键和实质性的差距存在于我们的知识， 理解分子动力学在蛋白质/DNA相互作用中的作用。一个普遍的问题， 分子识别领域的一个问题是，结构研究揭示了相对较少的熵成分的分子识别。 复合物形成的自由能。因此，通过以下方式补充结构信息非常重要： 进行研究，调查蛋白质/DNA界面的侧链动力学。 协同离子结合是钙信号通路的基本性质之一。的 细胞内钙信号的读出必须非常精细地调节以实现对瞬时的快速响应， 构成钙信号的Ca 2+浓度的细微变化。的重大意义 EF-hand CaBP与Ca 2+的协同结合促使人们努力确定Ca 2+的分子基础。 在这个蛋白质家族的特定成员的协同性。Calbindin D9 k是一种单结构域EF-手CaBP， 研究协同结合现象的主要模型系统。 本研究的基本假设是蛋白质侧链动力学的调节 在建立共有/非共有DNA之间的互补界面中起重要作用 序列和同源DNA结合蛋白，并在促进离子- 导致协同钙结合的结合位点。为了检验这些假设， 提出：（1）确定K50类分子的侧链动力学和热力学性质 来自人Pitx 2和果蝇Bicoid蛋白的同源结构域，与共有双链DNA结合 （2）确定Pitx 2和Bicoid同源结构域结合的结构、动力学和热力学 非共有DNA位点;（3）研究协同结合的分子基础和驱动力， CaBP钙结合蛋白D9 k对Ca ~（2+）的影响;（4）表征了侧链动力学在CaBP钙结合蛋白D9 k中的热力学作用。 端粒末端保护（TEP）蛋白的单链DNA结合家族。

The overall goal of this project is to investigate the contribution of amino acid side-chain dynamics to the physico-chemical mechanisms that determine the thermodynamics of recognition and association in protein/DNA interactions and the molecular basis of cooperativity of ion binding in calcium-binding proteins (CaBPs). Side chains can make significant contributions to the configurational entropy of a protein, thereby modulating the thermodynamics of protein function. A fundamental understanding of how proteins work therefore requires an intimate knowledge of the dynamic properties of the side chains. Two model systems, protein/DNA complexes and the CaBP calbindin D9k, have been selected for study that will allow key insights to be obtained regarding the role of side-chain dynamics in protein/DNA binding/recognition and cooperativity of ion binding, respectively. Despite the large number of structural and thermodynamic studies that have been reported for a variety of protein/DNA systems, critical and substantial gaps exist in our knowledge and understanding of the role played by molecular dynamics in protein/DNA interactions. A general problem in the field of molecular recognition is that structural studies reveal relatively little about the entropic component of the free energy of complex formation. Thus, it is very important to complement structural information by undertaking studies to investigate side-chain dynamics in the protein/DNA interface. Cooperative ion binding is one of the fundamental properties of calcium signaling pathways. The readout of intracellular calcium signals must be very finely tuned to effect a rapid response to the transient and subtle variations in Ca2+ concentrations that constitute the calcium signals. The great importance of cooperative binding of Ca2+ by EF-hand CaBPs has motivated efforts to determine the molecular basis for cooperativity in specific members of this protein family. Calbindin D9k, a single domain EF-hand CaBP, is one of the primary model systems for studying the cooperative binding phenomenon. The general hypotheses of the proposed research are that modulation of protein side-chain dynamics plays important roles in establishing a complementary interface between consensus/non-consensus DNA sequences and a cognate DNA-binding protein, and in promoting allosteric communication between ion- binding sites that leads to cooperative calcium binding. To test these hypotheses the following specific aims are proposed: (1) determine the side-chain dynamics and thermodynamic properties of the K50-class homeodomains from the human Pitx2 and the Drosophila Bicoid proteins, bound to a consensus duplex DNA site; (2) determine the structure, dynamics and thermodynamics of the Pitx2 and Bicoid homeodomains bound to non-consensus DNA sites; (3) investigate the molecular basis and driving forces for cooperative binding of Ca2+ by the CaBP calbindin D9k; and (4) characterize the thermodynamic role of side-chain dynamics in the single-strand DNA-binding family of Telomere End Protection (TEP) proteins.