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.

Project Narrative Environmental induced molecular structural changes in proteins and nucleic acids are essential for physiological processes of respiration and metabolism. The proposed research will follow the structural evolution of proteins and nucleic acids during their functionally relevant structural perturbations via high resolution X-ray snapshots of active sites and global protein conformations. Different stages of biological processes are triggered by external stimuli, e.g., ligand photodissociation, pH, temperature and reactant concentration jumps. These studies will ultimately advance our understanding of interplays among different structural factors and enable the control of protein/nucleic acid functions related to signal sensing, catalysis, and drug delivery.