| Studying the complex world of biology one molecule at a time allows for characterization of many indispensible mechanistic features of biochemical reactions that are otherwise invisible using ensemble based approaches. In this thesis I present two fluorescence-based single-molecule studies that investigate the reaction dynamics of an intricate biological process and contribute a new tool for single-molecule investigation.;First, following an overview of single-molecule Forster resonance energy transfer (smFRET), I report a smFRET study that monitors the remodeling dynamics of individual nucleosomes by the ATP-utilizing chromatin assembly and remodeling factor (ACF). The mechanism by which ACF mobilizes nucleosomes to generate regularly spaced nucleosome arrays required for heritable gene silencing and chromatin compaction remains poorly understood. Observation of ACF-catalyzed remodeling in real time by smFRET revealed previously unknown intermediates and dynamics of the process. In the presence of ACF and ATP, nucleosomes exhibit gradual translocation along DNA interrupted by well-defined kinetic pauses that occurred after approximately 7 or 3--4 base pairs of translocation. The binding of ACF, nucleosome translocation, and exiting of translocation pauses are all ATP-dependent, revealing three distinct functional roles of ATP during remodeling. At equilibrium, a continuously bound ACF complex can move a nucleosome back-and-forth many times before dissociation, indicating that ACF is a highly processive and bidirectional nucleosome translocase.;Secondly, I present a single-molecule fluorescence study which describes the discovery and characterization of a novel, all-optical single-molecule switch. The switch consists of two molecules: a primary fluorophore, the carbocyanine dye Cy5, that can be switched between a fluorescent and a dark state by light of different wavelengths, and a secondary fluorophore. Cy3, that facilitates switching. The interaction between the two molecules exhibits a distance-dependence much steeper than that of conventional FRET. This enables the switch to act as a spectroscopic ruler sensitive to distances difficult to access by other spectroscopic methods, thus presenting a new tool for the study for bio-molecules at the single-molecule level. Additionally, since its discovery, Cy5 photo-switching has enabled the emergence of new, super-resolution, fluorescence microscopy techniques that break the classical diffraction limit of light. |
