| Chapters 2-7 focus on physical enzymology. Despite its long history, recent single-molecule spectroscopy, among many others techniques, has generated new quantitative data that reveal unobserved features of protein dynamics and enzyme catalysis at unprecedented levels. Much of these are beyond the classic framework of transition state theory and Michalis-Menten (MM) enzyme kinetics. Due to the complexity of the problem, theoretical developments in this area have much lagged behind experiments.;After an initial experimental characterization on single-molecule protein conformational fluctuations, we then develop a dynamical rate theory for enzyme catalyzed chemical reactions, from a statistical mechanics approach. Towards this goal, we formulate a two-dimensional (2D) multi-surface free energy description of the entire catalytic process that explicitly combines the concept of "fluctuating enzymes" with the MM enzyme kinetics. The outcome of this framework has two folds. On the rate theory side, going much beyond transition state theory, it connects conformational fluctuations to catalysis, allows for the interplay between energetics (e.g. Haldane's stain energy) and dynamics (e.g. Koshland's induced fit), and predicts the time dependence of single-enzyme catalysis. On the enzyme kinetics side, it gives mechanistic and unified understanding of MM and non-MM (both positive and negative cooperativity) kinetics of monomeric enzymes, in term of non-equilibrium steady state cycle on the 2D free energy surface.;Chapters 8-11 present the principle and application of a new ultra-sensitive nonlinear optical microspectroscopy, femtosecond (fs) triple-resonant coherent anti-Stokes Raman scattering (CARS), in which the amplitude and phase of input fs laser pulses are optimally shaped to be in triple resonant with the molecular electronic and vibrational transitions to generate a coherent nonlinear signal beam at a new color with a highest possible efficiency. This technique combines the advantages of both coherent Raman scattering and electronic resonant Raman scattering within an optical microscopy, through the latest pulse shaping technology. We demonstrate the applicability of this technique on various different systems, including genetically encoded chromoproteins, and show that the current sensitivity is approaching 50 non-fluorescent molecules in aqueous solution with possible Raman spectral identification, thus boosting the sensitivity of the current CARS microscopy by 103 to 10 5 folds. |
