| The focus of this thesis is the development, verification, and scientific use of a force probe designed to explore weak interactions between or within single biomolecules. The developed system utilizes a laser optical trap to apply piconewton forces to a functionalized probe bead that interacts with a reactive test bead, and high-speed video processing to measure nanometer displacements of both beads at a rate of ∼1500 Hz. The position of the probe bead is used to report both the force on a single molecular bond and the change in length due to protein unfolding or refolding. Several feedback and automation systems have been integrated to facilitate the repetitive testing required to characterize these stochastic interactions.; The measurement and application of piconewton-sized forces depends on accurate position detection and proper calibration of the optical trap. The accuracy of the position detection was determined to be 2-3 nm by analyzing a non-moving bead. To assess optical trap calibration, a detailed study and comparison of common methods was performed. Noise-based methods which rely on the variance or power spectrum were found to have systematic errors due to the finite integration time of the detection. To solve this problem, the effect of integration time on these measurements was quantified and new calibration methods were developed.; Finally, the instrument was used to study the forced unfolding of the spectrin repeat, a small modular protein domain found in various load supporting proteins. The unfolding kinetics were determined by pulling on the molecule over several orders of magnitude in loading rate. The findings indicate that spectrin unfolding is governed by a single prominent energy barrier, characterized by a force scale of 2.5 pN and a stress-free unfolding rate of 0.003 s -1. |
