| Fluorescence spectroscopy has been extensively used to study biological molecules both in vivo and in vitro. Recent developments in single-molecule spectroscopy and imaging help to illustrate how each molecule works as a component of the network in real time. Using these techniques, two different systems have been studied in this thesis: the dynamics of single MreB protein in living Caulobacter crescentus cell, and the conformational changes of the chaperonin GroEL and its substrate, the von Hippel-Lindau tumor suppressor protein, VHL.; First, the motion of single fluorescent MreB-YFP fusions in living Caulobacter cells in a background of unlabeled MreB was measured and analyzed. Polymerized MreB and unpolymerized MreB monomers could be distinguished according to their motion, characterized as directional treadmilling and Brownian diffusion, respectively. Novel information was extracted about MreB filaments, including the rate of polymerization, as well as the average length and shape. These measurements suggested that the long MreB helical structure implied from bulk studies may be composed of short filaments bundles that lack a uniform global polarity.; Next, local conformational changes of the molecular chaperonin GroEL were studied with the polarity-sensitive fluorescence probe Nile Red. Nile Red was attached to the apical domain of a GroEL mutant (Cys261-NR). Bulk fluorescence spectra demonstrated that the conformational changes of Cys261-NR induced by substrate MDH, GroES and nucleotides could be followed by the fluorescence intensity of the Nile Red. The sequences of local conformational changes were studied with the addition of each reagent (substrate MDH, GroES, and various nucleotides) in different orders.; Lastly, structural changes of the substrate VHL during the folding cycle with the chaperonin GroEL/ES were probed by both ensemble and single-molecule FRET. These measurements allowed determination of how the VHL structure is adjusted after binding to chaperonin, followed by lid closure. Single-molecule FRET studies revealed that lid closure in GroEL induces further structural changes in various regions of VHL. These changes occurred heterogeneously right after the lid closure. No further significant structural adjustment occurred while VHL was encapsulated in the GroEL cavity, implying that multiple binding and releases to GroEL/ES are necessary for VHL folding. |
