Project Summary/Abstract Protein dynamics is essential for its biological function. With integration of molecular biology, state-of-the-art femtosecond spectroscopy and computation simulations, the biological processes now can be studied from the intial ultrafast dynamics to subsequent longtime motions on the most fundamental level and thus the molecular mechanisms can be revealed. We have recently investigated the dynamics and mechanisms of several biological photomachines such as photoenzymes and photoreceptors in nature. We mapped out the complete repair photocycles of UV-damaged thymine dimer in DNA by photoenzyme photolayses in real time, including ten steps of ultrafast elementary reactions, and reveled a unified electron-transfer molecular mechanism for photolyase superfamily. In another direction, we also made significant advances on the understanding of water-protein interactions and dynamics and elucidated the fundamental coupled motions between hydration water and protein sidechains on the picosecond time scales, providing direct envidence that hydration water controls sidechain fluctuations. The understanding of biological water is significant to a variety of biological activities such as protein recognition and enzymatic catalysis. In this new, synergistic effort, we take challenges to explore more new complex systems in three major areas: (1) investigating two photoenzymes of an intricate (6-4)-photoproduct photolyase and a newly discoivered fatty-acid photodecarboxylase to map out the entire enzymatic reactions and reveal complete catalytic photocycles. Both photoenzyems are significnat in nature to repair UV-damaged DNA and produce hydrocarbon biofuels; (2) examining three photoreceptors of UV-light UVR8, blue-light cryptochromes (DmCry and AtCry) and several red-light phytochromes to reveal the primary dynamics for initial signaling and subsequent conformational changes. The entire dynamic processes may occur from ultrafast femtoseconds to longtime milliseconds; (3) exploring further water-protein interactions and dynamics of complex biological systems for better understanding the role of water in protein structure, stability, dynamics and functions. We will systematically investigate the cavity-water dynamics in a giant chaperonin protein (GroEL) for understanding trapped water in function of substrate protein folding. We will add new powerful methods of the femtosecond x-ray free electron lasers (XFEL) technique and the high-level quantum mechanics/molecualr mechanics (QM/MM) calcualtions in these studies. We will develop new conceptes and make important discoveries. These frontiers we are pursuing will provide new knowledge for further biomedical applications.