NMR Studies of Protein Side-Chain Dynamics

描述（由申请人提供）：本项目的总体目标是研究氨基酸侧链动力学对确定蛋白质/DNA相互作用中识别和缔合热力学的物理化学机制的贡献，以及钙结合蛋白（CaBP）中离子结合协同性的分子基础。侧链可以对蛋白质的构型熵做出重要贡献，从而调节蛋白质功能的热力学。因此，对蛋白质如何工作的基本理解需要对侧链的动态特性有深入的了解。两个模型系统，蛋白质/DNA复合物和CaBP calbindin D9 k，已被选定的研究，将允许关键的见解，以获得关于蛋白质/DNA结合/识别和离子结合的协同性的侧链动力学的作用，分别。尽管大量的结构和热力学研究，已报告的各种蛋白质/DNA系统，关键和实质性的差距存在于我们的知识和理解的分子动力学在蛋白质/DNA相互作用中所发挥的作用。在分子识别领域的一个普遍问题是，结构研究揭示相对较少的熵的复杂形成的自由能的分量。因此，这是非常重要的，以补充结构信息进行研究，调查侧链动力学在蛋白质/DNA界面。协同离子结合是钙信号通路的基本性质之一。细胞内钙信号的读出必须非常精细地调节，以实现对构成钙信号的Ca 2+浓度的瞬时和细微变化的快速响应。合作的Ca 2+结合EF-手CaBP的重要性，激励了努力，以确定在这个蛋白质家族的特定成员的协同性的分子基础。Calbindin D9 k是一个单结构域EF-手型CaBP，是研究协同结合现象的主要模型系统之一。所提出的研究的一般假设是，蛋白质侧链动力学的调制在建立共识/非共识DNA序列和同源DNA结合蛋白之间的互补界面中起着重要作用，并在促进离子结合位点之间的变构通信，导致合作钙结合。为了验证这些假设，提出了以下具体目标：（1）确定来自人Pitx 2和果蝇Bicoid蛋白的K50类同源结构域与共有双链体DNA位点结合的侧链动力学和热力学性质：（2）确定Pitx 2和Bicoid同源结构域与非共有DNA位点结合的结构、动力学和热力学性质;（3）研究CaBP calbindin D9 k与Ca ~（2+）协同结合的分子基础和驱动力;（4）研究端粒末端保护蛋白（TEP）单链DNA结合家族中侧链动力学的热力学作用。公共卫生相关性：拟议的研究重点是提高我们对细胞功能至关重要的两个基本生物过程的理解：蛋白质/DNA结合/识别和合作离子结合。蛋白质/DNA复合物特别令人感兴趣，因为对指导结合和识别的原理的理解可以提出与DNA转录调控相关的许多医学和生物学问题的创新解决方案;基本的细胞活动，如转录，复制，重组和基因修复需要DNA和DNA结合蛋白的非共价相互作用。协同性是生物系统如钙结合蛋白家族的一个基本重要的功能特性;钙调节各种各样的细胞过程，如肌肉收缩、细胞周期控制、分化和信号转导，因此在人类健康中起着至关重要的作用。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The proposed research focuses on improving our understanding of two fundamental, biological processes that are critical for cellular function: protein/DNA binding/recognition and cooperative ion binding. Protein/DNA complexes are of particular interest because an understanding of the principles guiding binding and recognition could suggest innovative solutions to a number of medical and biological problems that are associated with the regulation of DNA transcription; fundamental cellular activities such as the transcription, replication, recombination and repair of genes require the non-covalent interaction of DNA and DNA-binding proteins. Cooperativity is a fundamentally important functional property of biological systems such as the family of calcium binding proteins; calcium regulates a wide variety of cellular processes, such as muscle contraction, cell-cycle control, differentiation and signal transduction, and thus plays an essential role in human health.