Protein Dynamics in Dihydrofolate Reductase Catalysis

二氢叶酸还原酶催化中的蛋白质动力学

基本信息

批准号：
8245745
负责人：
PETER Edwin WRIGHT
金额：
$ 38.32万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-04-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8245745
关键词：
Active Sites Address Adopted Adverse effects Anti-Bacterial Agents Anti-Infective Agents Antimalarials Antineoplastic Agents Antiprotozoal Agents Binding Catalysis Chemicals Chemistry Clinical Complement Complex Coupled Coupling Crystallography Data Development Dihydrofolate Reductase Enzymes Escherichia coli Event Evolution Folate Goals Grant Holoenzymes Human Kinetics Laboratory Research Location Measurement Measures Methods Methotrexate Modeling Molecular Molecular Conformation Molecular Machines Motion Movement NADP Pathway interactions Pharmaceutical Preparations Play Population Process Property Protein Dynamics Proteins Pyrimethamine Reaction Relative (related person)Relaxation Research Residual state Role Sampling Shapes Structure Testing Tetrahydrofolates Therapeutic Agents Thermodynamics Time Trimethoprim Vertebral column Work base cofactor design flexibility inhibitor/antagonist insight millisecond mutant novel pathogen protein structure public health relevance research study restraint sound theories three dimensional structure

项目摘要

DESCRIPTION (provided by applicant): Proteins are dynamic molecular machines, undergoing motions on a wide range of time scales. Although there is considerable evidence both from theory and experiment that many enzymes are inherently flexible, the fundamental question of how, or even if, protein fluctuations couple to catalytic function remains unanswered. Are protein motions coupled to the chemical transformation, or are they involved primarily in controlling the flux of substrate, products, or cofactors? What is the time scale of active site conformational changes required for catalysis? How is the energy landscape of the enzyme modulated during the catalytic cycle and how is it shaped during evolution? Are there species-related differences in protein dynamics and available conformational substates that might potentially be exploited for development of highly selective drugs that better discriminate between enzymes from humans and pathogens? These issues will be addressed using state-of-the-art NMR methods to elucidate the dynamic properties of an exceptionally well-characterized enzyme, dihydrofolate reductase (DHFR). DHFR is the target for anti-folate drugs such as the anticancer agent methotrexate and the antibacterials trimethoprim and iclaprim. The proposed research will focus on characterization of microsecond-millisecond time scale fluctuations in the active sites of E. coli and human DHFR, on the same time scale as key events in catalysis. NMR experiments will provide a detailed structural, dynamic, and thermodynamic description of slow motions of the active site loops in all intermediate states involved in the catalytic cycle and in carefully selected mutants that impair the catalytic process. These experiments build upon earlier research from this laboratory that showed how the energy landscape is modulated to funnel E. coli DHFR through its preferred catalytic pathway. The proposed research will provide novel insights into the energy landscapes of E. coli and human DHFR and will advance our understanding of the relationship between protein dynamics and catalytic function. PUBLIC HEALTH RELEVANCE: The proposed research will address the role of protein motions in controlling the catalytic function of the enzyme dihydrofolate reductase. This enzyme is of major clinical importance as a target for anticancer agents, anti-infectives, and anti-malarial drugs. The research will provide novel information on species-related differences in protein structure and dynamics that might potentially be exploited for development of highly selective drugs that better discriminate between enzymes from humans and pathogens to decrease undesirable side effects.

描述（由申请人提供）：蛋白质是动态分子机器，在很宽的时间尺度上进行运动。尽管理论和实验都有大量证据表明许多酶本质上是灵活的，但蛋白质波动如何或是否与催化功能耦合的基本问题仍未得到解答。蛋白质运动是否与化学转化相关，或者它们主要参与控制底物、产物或辅因子的通量？催化所需的活性位点构象变化的时间尺度是多少？酶的能量格局在催化循环过程中是如何调节的以及在进化过程中是如何形成的？蛋白质动力学和可用构象亚状态是否存在与物种相关的差异，这些差异可能被用来开发高选择性药物，以更好地区分人类酶和病原体酶？这些问题将使用最先进的 NMR 方法来解决，以阐明一种非常明确的酶二氢叶酸还原酶 (DHFR) 的动态特性。 DHFR 是抗叶酸药物（例如抗癌药物甲氨蝶呤以及抗菌药物甲氧苄啶和艾克拉普林）的靶标。拟议的研究将重点关注大肠杆菌和人类 DHFR 活性位点的微秒至毫秒时间尺度波动的表征，其时间尺度与催化中的关键事件相同。核磁共振实验将提供催化循环中涉及的所有中间状态以及精心选择的损害催化过程的突变体中活性位点环的慢运动的详细结构、动力学和热力学描述。这些实验建立在该实验室早期研究的基础上，该研究展示了如何调节能量景观以通过其首选的催化途径引导大肠杆菌 DHFR。拟议的研究将为大肠杆菌和人类 DHFR 的能量景观提供新的见解，并将增进我们对蛋白质动力学和催化功能之间关系的理解。公共健康相关性：拟议的研究将解决蛋白质运动在控制二氢叶酸还原酶催化功能中的作用。这种酶作为抗癌剂、抗感染剂和抗疟疾药物的靶标具有重要的临床重要性。该研究将提供有关蛋白质结构和动力学方面与物种相关的差异的新信息，这些信息可能用于开发高选择性药物，更好地区分人类酶和病原体酶，以减少不良副作用。