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

圣安东尼奥，美国德克萨斯大学健康科学中心的琳达·罗曼博士获得了化学系生命过程化学项目的支持，获得了国际化学合作奖（ICC），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研究机构的支持。凭借该奖项，生命过程的化学计划正在资助圣安东尼奥的德克萨斯大学健康科学中心的琳达罗曼博士和威斯康星州医学院的JungJaKim博士，以开发用于一氧化氮脱氢酶机制的探针并进行该机制的研究。 一氧化氮合酶（nitricoxidetransistases，NOS）产生一氧化氮（nitricoxide，NO），在许多生理过程中起关键作用。内皮型一氧化氮合酶（eNOS）在心血管系统中产生NO，在那里它是一种有效的血管扩张剂。 类似地，神经元同种型（nNOS）参与神经传递以及骨骼肌和心肌功能，并且诱导型NOS（iNOS）响应于免疫攻击而表达。 研究人员将使用定制的光活化探针研究这些酶的功能机制。调节这些酶的活性的能力将有助于阐明它们的生理作用和调节，并深入了解它们的机制。 这一追求将使本科生和博士后获得蛋白质表达、纯化、表征以及使用X射线衍射确定蛋白质结构方面的专业培训。 该项目的重点是使用新型的可光活化的NADPH类似物，称为纳米触发物（NTs），靶向一氧化氮合酶（NOS）亚型和细胞色素P450还原酶（POR）的NADPH位点，阐明通过这些酶的电子转移机制，以及稳定它们在一个封闭的构象，促进结晶。该项目将：（1）确定各种NT-蛋白质复合物的X射线结构;（2）通过溶液动力学研究和时间分辨X射线晶体学阐明第一催化步骤的时间分辨结构/功能研究;（3）通过计算机模拟设计新型亚型特异性eNOS激活剂;和（4）由于NT探针的固有成像特性和特异性，通过双光子激发监测细胞中的eNOS运输。待开发和表征的光活性工具可以在用激光脉冲照射时触发特定的催化事件。激光脉冲发生在零时间，允许催化剂的启动同步，这可以以时间分辨的方式进行监测。该提案将开发用于选定蛋白质的时间分辨研究的新工具，这些工具通过直接结合蛋白质来克服探针缓慢（大于或等于ms）扩散的限制，并能够通过超快电子注入蛋白质来触发催化。这种方法代表了一种新的工具，以同步在溶液中的酶诱导的激光脉冲的合奏，并代表了一种替代单分子研究。

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.