| The focus of this thesis is the development and application of a novel technique for investigating the structure and dynamics of weak interactions between and within single-molecules. This approach is designed to explore unusual features in bi-directional transitions near equilibrium. The basic idea is to infer molecular events by observing changes in the three-dimensional Brownian fluctuations of a functionalized microsphere held weakly near a reactive substrate. Experimentally, I have developed a unique optical tweezers system that combines an interference technique for accurate 3D tracking (∼1 nm vertically, and ∼2-3 nm laterally) with a continuous autofocus system which stabilizes the trap height to within 1-2 mn over hours. A number of different physical and biological systems were investigated with this instrument. Data interpretation was assisted by a multi-scale Brownian Dynamics simulation that I have developed. I have explored the 3D signatures of different molecular tethers, distinguishing between single and multiple attachments, as well as between stiff and soft linkages. As well, I have developed a technique for measuring the force-dependent compliance of molecular tethers from thermal noise fluctuations and demonstrated this with a short ssDNA oligomer. Another practical approach that I have developed for extracting information from fluctuation measurements is Inverse Brownian Dynamics, which yields the underlying potential of mean force and position dependent diffusion coefficient from the Brownian motion of a particle. I have also developed a new force calibration method that takes into account video motion blur, and that uses this information to measure bead dynamics. Perhaps most significantly, I have trade the first direct observations of the refolding of spectrin repeats under mechanical force, and investigated the force-dependent kinetics of this transition. |
