Capturing Transient Protein and Nucleic Acid Structures During Their Functions on Multiple Spatial and Temporal Scales

Summary/Abstract The long term objective of the proposed research is to develop an integrated instrumentation capable of studying protein/nucleic acid structural dynamics that are relevant to their functions on the time scales from femtosecond to millisecond in order to gain new insight into correlations of active site structures and global conformations of these molecules. Snapshots of solution phase molecular structures over different spatial scales, from sub-Ångström for active sites to several nanometers for overall conformation, will be captured using time-resolved X-ray spectroscopy and scattering. These structural studies will be combined with advanced molecular dynamics simulations that will generate detailed atomistic dynamics consistent with measured scattering profiles over a wide-range of temporal scales from femtosecond to millisecond. The proposed research is complementary to single crystal X-ray diffraction, and intends to map reaction trajectories through three-dimensional structures as a function time in media that mimic biological environments. In order to detect structural changes in an ensemble, reaction triggers must be designed to create sudden environmental changes that synchronize actions of the molecules with much higher time resolution than traditional mixing. The program has three main innovations from previous studies: 1) to develop triggering sources beyond direct light excitation used in the past to initiate reactions to overcome the limitation that very few biological systems related to human health are light activated for their function; 2) to develop novel sample delivery system that reduces the sample consumption by a factor of 100 and enables many precious laboratory samples to be studied using the time-resolved X-ray methods; and 3) to develop a combined approach in data analyses using advanced molecular dynamics simulation coupled to time-dependent X-ray scattering data to extract structures with improved structural accuracy especially for those coexisting species. The above innovation in methodology will allow us to investigate a number of systems that are biologically significant for enzymatic reactions, signal sensing, protein/nucleic acid folding/unfolding as well as lipids phase transitions. Several systems are chosen for the proposed studies to capture transient structures of, a) local metal center and global protein conformations of cytochrome c oxidase model proteins triggered by photodissociation of inhibitors; b) protein folding induced by calcium ion a concentration jump; c) temperature-induced RNA conformational changes sensing signal for translation; d) pH-dependent DNA structures for human oncogene regulation and e) pH-responsive lipid nanocarrier assembly for anticancer drug delivery. These structural results combined with those of reaction kinetics from optical transient spectroscopy will provide guidance for modulating protein and nucleic acid functions via structural modifications, which will lead to impacts in drug design, enzymatic function enhancement, catalysis, as well as theoretical calculations.

摘要/摘要 拟议研究的长期目标是开发能够的综合仪器 研究与其功能在时间尺度上相关的蛋白质/核酸结构动力学 从飞秒到毫秒到毫秒，以便获得有关活动现场结构和相关性的新见解 这些分子的全球构象。溶液相分子结构的快照 空间量表，从积极部位的子Ångström到多种纳米的整体构象，将是 使用时间分辨的X射线光谱和散射捕获。这些结构研究将合并 通过高级分子动力学模拟，将产生详细的原子动力学一致 从飞秒到毫秒的临时尺度上进行了测量的散射曲线。 所提出的研究是单晶体X射线衍射完整的，并打算绘制反应 通过三维结构作为模拟生物学的媒介中的功能时间的轨迹 环境。为了检测合奏中的结构变化，反应触发器必须设计为 突然创建环境变化，使分子的动作与更高的时间同步 分辨率比传统混合。该计划有以前研究的三个主要创新：1） 开发触发源超出了过去用于发起反应以克服的直接光兴奋 很少有与人类健康相关的生物系统的功能激活的局限性。 2）开发新型样品输送系统，将样品消耗降低100倍，并且 使用时间分辨的X射线方法可以使许多贵重的实验室样品进行研究；和3）到 使用高级分子动力学模拟在数据分析中开发一种组合方法 时间依赖于X射线散射数据，以提高结构准确性，尤其是提取结构 对于那些共存的物种。上述方法论的创新将使我们能够调查许多 对于酶促反应，信号感应，蛋白质/核酸具有生物学意义的系统 折叠/展开以及脂质相变。选择了几个系统进行拟议的研究 为了捕获瞬态结构，a）局部金属中心和细胞色素c的全局蛋白质构象 氧化酶模型蛋白是由抑制剂的光解离引发的； b）钙诱导的蛋白质折叠 离子浓度跳跃； c）温度引起的RNA构象变化灵敏度信号 翻译; d）人类癌基因调节和e）pH响应性脂质的pH依赖性DNA结构 用于抗癌药物输送的纳米载体组件。这些结构性结果与反应的结果相结合 光学瞬态光谱的动力学将为调节蛋白质和核酸的指导提供指导。 通过结构修饰的功能，这将导致药物设计的影响 增强，催化以及理论计算。