Biology's wires and motors : single-molecule mechanics of M13 bacteriophage and kinesin

Nature serves as an inspiration for engineering design, and, conversely, engineering principles have helped to usher in a quantitative frontier of biology. This intricate interdependency between engineering and biology is strongly evident in the single-molecule regime, where high-resolution tools are addressing previously intractable biological problems, while nanotechnology is being accelerated by advances in molecular biology. This thesis explores both sides of the dichotomy in an effort to reveal the mechanics of two biological systems M13 filamentous bacteriophage and kinesin motor protein and the properties of their underlying parts. M113 is a filamentous virus that has elicited the interest of the engineering community for its use in phage display of combinatorial peptide libraries and more recently as a 1D template for organizing and growing inorganic materials. Both applications rely on the direct link between genotype and phenotype, whereby each phage particle displays fusion molecules on its proteinaceous capsid and simultaneously carries the encoding DNA. A better understanding of its polymer mechanics is critical to the further development of future M13-based technologies. As a result, combined efforts in genetic engineering, optical trapping, and modeling were employed in the first characterization of the biopolymer's single-molecule elasticity, revealing a persistence length (1265 nm) that places it squarely in the semiflexible polymer regime. Single-molecule stretching also revealed a mechanically robust and genetically versatile tether that could serve as a general "molecular handle" for single-molecule biophysics. As such, M13 was further engineered to incorporate variants of the zinc finger, DNA-binding domain of transcription factor Zif268 as minor coat fusions. Single-molecule assays centered around the "zinc fingered-phages" are being developed to establish M13 as a standalone, single-molecule building block for achieving universal connectivity and for unlocking future studies on the nature of protein-DNA interactions. Kinesin is an ATPase that "walks" processively along biofilament tracks to perform vital cellular processes. Whereas M13 mechanics were studied in the context of classical rod or polymer bending, our focus within kinesin mechanics is the molecular aspects of motility. While significant progress has been made in elucidating the broad features of the kinesin mechanochemical cycle, details of the force generation mechanism remain a mystery. Combined efforts in molecular biology, optical trapping, and molecular simulation were employed to put forth a novel mechanism for the motor's power stroke, namely that it is produced when the conserved N-terminal cover strand forms a a-sheet with the neck linker to yield the cover-neck bundle. In agreement with simulation, single-molecule motility data revealed impairment of the force-generating capacity of cover strand mutants, as measured by a reduction in stall force from the wild-type. Motility data also suggest that targeting forcegenerating elements, such as the cover strand, is a plausible strategy for designing biological motors with tunable motile properties, bringing us one step closer to a complete blueprint of kinesin's parts and their collective functioning. Thesis Supervisor: Angela M. Belcher Title: Germehausen Professor of Materials Science & Engineering, and Biological Engineering Thesis Supervisor: Matthew J. Lang Title: Associate Professor of Mechanical Engineering, and Biological Engineering