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，在广泛的时间尺度上进行动作。尽管从理论和实验中都有大量证据表明许多酶本质上是灵活的，但蛋白质波动如何催化功能的基本问题仍未得到解答。蛋白质运动是否与化学转化相结合，还是主要控制底物，产物或辅因子的通量？催化所需的主动部位构象变化的时间尺度是什么？在催化周期期间，酶的能量景观如何调节？是否存在与物种相关的蛋白质动力学和可用构象取代的差异，这些构象取代可能会被利用用于开发高度选择性的药物，从而更好地区分酶与人类和病原体？这些问题将使用最先进的NMR方法解决，以阐明特征良好的酶，二氢叶酸还原酶（DHFR）的动态特性。 DHFR是抗叶酸药物的靶标，例如抗癌剂甲氨蝶呤和抗菌甲甲氧甲非甲甲胺和iClaprim。拟议的研究将集中于与大肠杆菌和人类DHFR活跃部位的微秒毫秒毫秒时尺度波动的表征，与催化中的关键事件相同。 NMR实验将提供详细的结构，动态和热力学描述，以描述参与催化循环的所有中间状态以及损害催化过程的精心选择的突变体中的活性位点环的缓慢运动。这些实验基于该实验室的早期研究，该研究表明了如何通过其首选的催化途径调节能量景观以漏斗大肠杆菌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，并将促进我们对蛋白质动力学与催化功能之间关系的理解。公共卫生相关性：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方面的作用。作为抗癌剂，抗感染药物和抗疟疾药物的靶标，该酶具有主要的临床重要性。这项研究将提供有关物种相关蛋白质结构和动力学差异的新信息，这些信息可能被利用，这些信息可能被利用用于开发高度选择性的药物，这些药物可以更好地区分人类和病原体的酶，以减少不良的副作用。