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

描述(申请人提供)：酶驱动的催化控制着所有生物体内的大多数代谢途径，但人们对此知之甚少。酶失活会导致遗传性疾病，如苯丙酮尿症，以及癌症，如着色性干皮病。尽管在小分子和抗体催化方面取得了很大进展，但由于缺乏对自然产生的酶的显著特异性和催化能力的了解，药物设计工作一直受到阻碍。我们的目标是开发一种全新的结构技术，基于世界上第一台X射线自由电子激光(XFEL)，以前所未有的结构分辨率跟踪非晶体样品的酶构象变化。由SLAC国家加速器实验室开发的XFEL作为直线加速器相干光源或LCLS，可发射持续数十飞秒(10-15秒)的X射线脉冲，并在单个脉冲中提供约2MJ的能量。激光已经显示出了获取生物分子纳米晶体数兆字节结构信息的能力。目前的建议是对生物分子溶液的非晶态液滴进行测量，在这种非晶态下，酶分子可以在接近生理条件下经历其催化循环。酶和底物的快速混合之后，将被注入到X射线激光光束中，给出一系列酶构象变化的快照，这将有助于设计能够干预酶功能的药物分子。通过将测量结果与对酶循环中被激活的结构中间体的计算机模拟相结合，我们将能够建立催化功能的路线图，这将提供一个自由能景观来指导酶工程和药物设计工作。这将使我们能够获得以前无法获得的关于亲环素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在人类健康中的两个最重要的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点。艾滋病毒已被证明在其病毒复制的最后阶段使用人类亲环素。

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.