Biology's wires and motors : single-molecule mechanicsof M13 bacteriophage and kinesin; Single-molecule mechanics of M13 bacteriophage andkinesin

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

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. M13 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 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. Com3 bined 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 [beta]-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…