International Collaboration in Chemistry: Time-resolved Studies of Endothelial Nitric Oxide Synthase Catalytic Mechanism Using Photoactive NADPH Analogues

美国圣安东尼奥德克萨斯大学健康科学中心的琳达·罗曼博士得到了化学系生命过程化学项目的支持，获得了国际化学合作(ICC)奖，该奖项包括与Anny Slama-Schwok博士、Joanne Xie教授和Eric Deprez博士的国际合作，后者将得到法国国家统计局的支持。生命过程化学计划资助德克萨斯大学圣安东尼奥健康科学中心的琳达·罗曼博士和威斯康星医学院的Jung Ja Kim博士开发一氧化氮合酶的探测器并对其机制进行研究。一氧化氮合酶(NOS)产生一氧化氮(NO)，在许多生理过程中起着关键作用。内皮型一氧化氮合酶(ENOS)在心血管系统中产生NO，是一种有效的血管扩张剂。同样，神经元异构体(NNOS)参与神经传递、骨骼肌和心肌功能，诱导型一氧化氮合酶(INOS)在免疫应答中表达。研究人员将使用定制的可光激活探针来研究这些酶的作用机制。调节这些酶活性的能力将有助于阐明它们的生理作用和调节，以及深入了解它们的机制。这项研究将允许本科生和博士后在蛋白质的表达、纯化、表征以及使用X射线衍射来确定蛋白质结构方面获得专门的培训。与NOSS结合的特定化合物也将被设计来帮助实现这些目标，使受训者接触合成化学技术。这个项目的重点是使用新型的可光激活的NADPH类似物，称为纳米触发剂(NTS)，针对一氧化氮合酶(NOS)异构体和细胞色素P450还原酶(POR)的NADPH位点，以阐明通过这些酶的电子转移的机制，并将它们稳定在封闭的构象中，促进结晶。该项目将：(1)确定各种NT-蛋白质复合体的X射线结构；(2)通过溶液动力学研究和时间分辨X射线结晶学，阐明第一步催化步骤的时间分辨结构/功能研究；(3)通过电子模拟设计新型的同型eNOS激活剂；(4)利用NT探针的固有成像特性和特异性，通过双光子激发监测eNOS在细胞中的运输。待开发和表征的光活性工具可以在激光脉冲照射下触发特定的催化事件。激光脉冲发生在零时间，允许催化启动的同步，这可以以时间分辨的方式进行监控。这一提议将为选定蛋白质的时间分辨研究开发新的工具，这些工具通过直接与蛋白质结合来克服探针缓慢(大于或等于ms)扩散的限制，并能够通过向蛋白质注入超快电子来触发催化。这种方法代表了一种新的工具来同步溶液中由激光脉冲诱导的酶系集，并代表了单分子研究的另一种选择。

Dr. Linda Roman at the University of Texas Health Science Center, San Antonio, USA, is supported by the Chemistry of Life Processes Program in the Division of Chemistry, for an International Collaboration in Chemistry (ICC) award that comprises an international collaboration with Dr. Anny Slama-Schwok, Professor Joanne Xie and Dr. Eric Deprez, who will be supported by Agence Nationale de la Recherche in France.With this award, the Chemistry of Life Processes Program is funding Dr. Linda Roman from the University of Texas Health Science Center in San Antonio and Dr. Jung Ja Kim from the Medical College of Wisconsin to develop probes for and undertake an investigation of the mechanism of nitric oxide synthases. The nitric oxide synthases (NOS) generate nitric oxide (NO), which plays key roles in many physiological processes. The endothelial nitric oxide synthase (eNOS) generates NO in the cardiovascular system, where it is a potent vasodilator. Similarly, the neuronal isoform (nNOS) is involved in neurotransmission and skeletal and cardiac muscle function, and the inducible NOS (iNOS) is expressed in response to an immune challenge. The investigators will study the mechanism of function of these enzymes with the use of customized photoactivatable probes. The ability to modulate the activities of these enzymes will help elucidate their physiological roles and regulation, as well as give insight into their mechanisms. This pursuit will allow undergraduate students and postdoctoral fellows to acquire specialized training in protein expression, purification, characterization, and in the use of X-ray diffraction to determine the protein structure. Specific compounds that bind to the NOSs will also be designed to help achieve these goals, exposing trainees to synthetic chemistry techniques.This project focuses on the use of novel photoactivatable NADPH analogues, called nanotriggers (NTs), targeted to the NADPH site of nitric oxide synthase (NOS) isoforms and cytochrome P450 reductase (POR), to elucidate the mechanism of electron transfer through these enzymes as well as stabilize them in a closed conformation, facilitating crystallization. This project will: (1) determine the X-ray structure of various NT-protein complexes; (2) elucidate time-resolved structure/function studies of the first catalytic steps by kinetics studies in solution and time-resolved X-ray crystallography; (3) design novel isoform-specific eNOS activators by in silico simulations; and (4) monitor eNOS trafficking in cells by biphotonic excitation, due to the intrinsic imaging properties and specificity of the NT probes. The photoactive tools to be developed and characterized can trigger a specific catalytic event upon irradiation with a laser pulse. The laser pulse occurs at zero time, allowing for synchronization of initiation of catalysis, which can be monitored in a time-resolved manner. This proposal will develop new tools for time-resolved studies of selected proteins that overcome the limitation of slow (greater than or equal to ms) diffusion of the probe by binding directly to the protein with the ability to trigger catalysis by ultrafast electron injection to the protein. This approach represents a new tool to synchronize an ensemble of enzymes in solution induced by a laser pulse and represents an alternative to single molecule studies.