Tracking conformational states in enzyme catalysts using correlated X-ray scatter

描述（由申请人提供）：酶驱动的催化控制着所有生物体中的大多数代谢途径，但对其了解甚少。酶的失效导致遗传疾病，如苯丙酮尿症和癌症，如色素性干皮病。尽管在小分子和抗体催化方面取得了很大进展，但由于缺乏对天然存在的酶的显著特异性和催化能力的了解，药物设计的努力受到阻碍。我们的目标是开发一种基于世界上第一个x射线自由电子激光器（xFEL）的全新结构技术，以前所未有的结构分辨率跟踪非晶体样品的酶构象变化。在SLAC国家加速器实验室作为直线加速器相干光源开发的xFEL，或LCLS提供持续几十飞秒（10-15秒）的x射线脉冲，并在单脉冲中提供约2mJ的能量。激光已经显示出它获得生物分子纳米晶体结构信息的能力。目前的建议是对非晶体形式的生物分子溶液的液滴进行测量，酶分子可以在接近生理条件下进行催化循环。酶和底物的快速混合之后，将注射到x射线激光束中，给出一系列酶构象变化的快照，这将有助于设计可以干预酶功能的药物分子。通过将测量结果与在酶循环过程中被激活的结构中间体的计算机模拟相结合，我们将能够构建催化功能的路线图，这将为指导酶工程和药物设计工作提供自由能景观。这将使我们能够获得以前无法获得的关于亲环蛋白A （CypA）功能的信息，CypA是蛋白质折叠，信号转导，运输，受体组装，细胞周期调节和应激反应的核心酶。CypA在人类健康中最重要的两个作用是控制免疫抑制和病毒感染。CypA是广泛使用的免疫抑制剂环孢素的靶标。HIV病毒已被证明在病毒复制的最后阶段使用人类亲环蛋白。

DESCRIPTION (provided by applicant): Enzyme-driven catalysis controls most metabolic pathways in all living organisms, yet is poorly understood. Enzymatic failures lead to genetic disease, such as phenylketonuria, and cancers such as xeroderma pigmentosum. Although much progress has been made in small molecule and antibody catalysis, drug design efforts have been impeded by lack of understanding of the remarkable specificity and catalytic power of naturally occurring enzymes. We aim to develop an entirely new structural technique based on the world's first X-ray free electron laser (xFEL), to track enzyme conformational changes with unprecedented structural resolution for non-crystalline samples. The xFEL, which has been developed at the SLAC National Accelerator lab as the Linac Coherent Light Source, or LCLS delivers pulse of x-rays which last a few tens of femtoseconds (10-15 seconds) and deliver about 2mJ of energy in a single pulse. The laser has already shown its ability to obtain terabytes of structural information on nano crystals of biomolecules. The present proposal is to do measurements on droplets of solution of biomolecules in non-crystalline form in which enzyme molecules can go through their catalytic cycles under close to physiological conditions. Rapid mixing of enzyme and substrate will be followed by injection into the x-ray laser beam giving a series of snapshots of changes in conformation of the enzyme which will help in design of drug molecules which can intervene in the functioning of the enzyme. By combining the measurements with computer simulations of the structural intermediates which are activated during the enzymatic cycle, we will be able to build a roadmap of the catalytic function that will provide a free energy landscape to guide enzyme engineering and drug design efforts. This will enable us to obtain previously inaccessible information on the function of Cyclophilin A (CypA), an enzyme central to protein folding, signal transduction, trafficking, receptor assembly, cell cycle regulation and stress response. Two of CypA's most important roles in human health are in controlling immunosuppression and viral infection. CypA is the target of the widely used immunosuppressive cyclosporine. The HIV virus has been shown to use human cyclophilin during its final stages of viral replication.