Conformational Motions of DHFR during Catalysis

催化过程中 DHFR 的构象运动

• 批准号: 6938201
• 负责人: NINA M GOODEY
• 金额: $ 4.4万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-15 至 2007-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6938201
• 关键词: bioimaging /biomedical imaging; conformation; dihydrofolate reductase; enzyme mechanism; fluorescence resonance energy transfer; molecular /cellular imaging; molecular dynamics; postdoctoral investigator

DESCRIPTION (provided by applicant): It is suspected that an enzyme's conformational motions may correlate closely to its catalytic function. Appreciation of how protein conformational fluctuations relate to catalysis will have profound implications for drug design and protein engineering efforts. The current proposal is centered on the implementation of novel single molecule fluorescence resonance energy transfer (FRET) methods to probe the dynamic motions of dihydrofolate reductase from Escherichia coli (DHFR), during catalysis. Two probes will be placed on DHFR, one in a stationary loop and the other on the Met20 loop, which undergoes conformational changes in the course of the DHFR catalytic cycle. The distance separating the tags will vary depending on the conformation of the moving Met20 loop. Single DHFR molecules will be attached to a glass slide via a biotinylated N-terminus. The conformational motions of the Met20 loop in a single DHFR molecule can thus be monitored during catalysis (equilibrium conditions) on the millisecond time-scale by changes in FRET efficiency, which is sensitive to the distance changes between the fluorescent tags. DHFR is an important target of several anticancer and antibacterial drugs and progress toward understanding its conformational fluctuations during catalysis will contribute to the design of new DHFR inhibitors that can bind to areas other than the active site to effectively target human diseases.

描述（由申请人提供）： 人们怀疑酶的构象运动可能与其催化功能密切相关。了解蛋白质构象波动与催化作用的关系将对药物设计和蛋白质工程的研究产生深远的影响。目前的建议是集中在实施新的单分子荧光共振能量转移（FRET）的方法来探测从大肠杆菌（DHFR）的二氢叶酸还原酶的动态运动，在催化过程中。将两个探针置于DHFR上，一个在固定环中，另一个在Met 20环上，其在DHFR催化循环过程中经历构象变化。标签之间的距离将根据移动的Met 20环的构象而变化。单个DHFR分子将通过生物素化的N-末端连接到载玻片上。因此，可以通过FRET效率的变化在催化（平衡条件）期间在毫秒时间尺度上监测单个DHFR分子中Met 20环的构象运动，FRET效率对荧光标签之间的距离变化敏感。DHFR是几种抗癌和抗菌药物的重要靶点，了解其在催化过程中的构象波动将有助于设计新的DHFR抑制剂，这些抑制剂可以结合到活性位点以外的区域，以有效地靶向人类疾病。