NMR Studies of Protein Side-Chain Dynamics

描述(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决定蛋白质/DNA相互作用中识别和结合热力学的物理化学机制的贡献，以及钙结合蛋白(CaBP)离子结合协作性的分子基础。侧链可以对蛋白质的构型熵做出重大贡献，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性质有深入的了解。选择了两个模型体系--蛋白质/DNA复合体和CABP calbindin D9k进行研究，这将使我们能够获得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的协同性的关键见解。尽管已经报道了大量关于蛋白质/DNA体系的结构和热力学研究，但在我们对分子动力学在蛋白质/DNA相互作用中所扮演的角色的认识和理解方面存在着关键的和实质性的差距。分子识别领域的一个普遍问题是，结构研究揭示的关于络合物形成自由能的熵分量相对较少。因此，通过研究蛋白质/DNA界面的侧链动力学来补充结构信息是非常重要的。协同离子结合是钙信号通路的基本性质之一。细胞内钙信号的读数必须非常精细地调整，以对构成钙信号的钙离子浓度的瞬时和细微变化做出快速反应。EF-Hand CaBP协同结合钙离子的重要性促使人们努力确定该蛋白家族特定成员协同作用的分子基础。Calbindin D9k是一个单区EF-Hand CABP，是研究协同结合现象的主要模型系统之一。这项研究的一般假设是，蛋白质侧链动力学的调节在建立共识/非共识DNA序列与同源DNA结合蛋白之间的互补界面以及促进离子结合部位之间的变构通讯从而导致协同钙结合方面发挥着重要作用。为了验证这些假说，我们提出了以下具体目标：(1)确定与一致的双链DNA位点结合的人Pitx2和果蝇双核样蛋白K50-类同源结构域的侧链动力学和热力学性质；(2)确定与非共识DNA位点结合的Pitx2和双链同源结构域的结构、动力学和热力学；(3)研究CABP calbindin D9k与钙离子协同结合的分子基础和驱动力；以及(4)表征侧链动力学在端粒末端保护蛋白单链DNA结合家族中的热力学作用。公共卫生相关性：拟议的研究重点是提高我们对两个对细胞功能至关重要的基本生物学过程的理解：蛋白质/DNA结合/识别和合作离子结合。蛋白质/DNA复合体之所以特别令人感兴趣，是因为对指导结合和识别的原理的理解可以为与DNA转录调控相关的许多医学和生物学问题提出创新的解决方案；基本的细胞活动，如基因的转录、复制、重组和修复，需要DNA和DNA结合蛋白的非共价相互作用。协作性是生物系统的重要功能特性，如钙结合蛋白家族；钙调节多种细胞过程，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.