| This thesis focuses on experimental and computational studies of spontaneous and functionally related conformational changes in protein molecules at the single-molecule level. Several technical advances were made in detecting subtle conformational changes within a protein molecule using methods such as electron transfer and spatial hindrance. These methods were used to study three major problems.; First, the electron transfer from a Tyr residue to the nearby flavin chromophore was used as a probe to study the spontaneous conformational fluctuation in a single NADH: flavin oxidoreductase molecule. The observation of pair-wise distance fluctuations on the angstrom scale led to the discovery of a highly stretched autocorrelation function for distance fluctuations, which suggests protein conformational fluctuations take place over at least four decades of time scales, from 1 ms to ∼ 10s. Molecular dynamics simulation on the same system also showed non-exponential distance fluctuations on much shorter time scale (nanoseconds) accessible by simulation, analogous to the experimental results on a longer time scale. Furthermore, simulations performed at different temperatures suggest that the complex dynamics have an energetic (rather than entropic) origin due to barrier crossing processes on the rugged potential energy landscape.; Second, as a model system for the study of proton-transfer reaction at the single-molecule level, we investigated spontaneous proton-transfer of a pH indicator SNARF, which has different emission spectra for different protonation states. In a two-color single-molecule fluorescence measurement, a sub-millisecond spectral fluctuation of individual SNARF molecule was observed when the molecule was in rapid equilibrium between its protonation states.; Third, the spatial hindrance of a fluorescent Cy3 molecule was used to study a functionally important conformational change of T7 DNA polymerase during catalysis. Upon binding of the substrate dNTP, the polymerase-DNA complex undergoes a conformational change, which was detected by an increase of fluorescence intensity of a single Cy3 labeled DNA molecule. This conformational change corresponds to an open-to-close motion of the fingers subdomain of the polymerase. We found the rate of the conformational change is sensitively dependent on whether the incoming dNTP is complimentary to the DNA template, and conclude that this conformational change plays a major role in substrate selection, which is pertinent to the fidelity of DNA replication catalyzed by DNA polymerases. |
