- Restrictions on Access:
- Open Access.
- Kinesins are motor proteins that perform essential cellular functions such as intracellular transport along microtubules and the organization of mitotic spindle during cell division. The structure of kinesin-1 consists of two heads attached through flexible neck-linkers to a coiled-coil stalk that ends in a cargo-binding domain. Kinesin-1 derives energy from ATP hydrolysis and walks in a hand-over-hand manner with each head taking 16-nm steps along the microtubules. In published work from the Hancock lab, single molecule experiments were performed to understand the mechanochemical transitions that underlie kinesin stepping by attaching 30 nm gold nanoparticle to one of the two heads of kinesin-1 heads through a 14 amino acid Avi-tag. Using Interferometric Scattering or Dark Field Total Internal Reflection Microscopy, millisecond temporal resolution and 1 nm spatial precision were achieved in this work. Similar experiments that showed somewhat different behavior were performed by the Tomishige lab, using a PEG-tag, which is shorter and less elastic tether than an Avi-tag, and was attached at a different location on the head. Interpreting these measurements taken at millisecond timescales requires a more detailed understanding of the microsecond-scale diffusion of the kinesin head and coupled nanoparticle. Specifically, it is important to understand how the attached nanoparticle affects the dynamics of the head and whether the nanoparticle faithfully tracks the head position. To address these questions, the present study used Brownian Dynamics modelling to simulate the three-dimensional dynamics of a 30-nm nanoparticle tethered to a kinesin-1 head via either an Avi-tag or PEG-tag. In the two-head-bound state, a nanoparticle tethered by an Avi-tag tracked the head more accurately along the axis of the microtubule than a particle tethered through a PEG-tag, but tracking accuracy perpendicular to the microtubule were identical for the two tethers. In the one-head-bound state, both heads tracked nanoparticles with similar accuracy, but the PEG-tag created a larger force in the neck-linker domains than did the Avi-tag. According to these data, an Avi-tag is a better tag for tracking the head, as it more accurately tracks head position and creates less perturbation in the natural system than a PEG-tag. To better study the effects of different experimental parameters on nanoparticle tracking accuracy, a simpler model consisting of a nanoparticle tethered to a glass surface was used. In the absence of added experimental noise, particle size and contour length of the tether were found to have major effects on tracking accuracy, defined as the Root-Mean-Squared (RMS) error between imaged and true particle position, but the persistence length had only a minor influence. With simulated experimental noise added, the Avi-tag and PEG-tags gave similar RMS error of tracking, demonstrating that noise inherent in the imaging process had a larger effect on the measured particle position than did the mechanical properties of the tether. Kinesins are implicated in neurodegenerative diseases and are targets for anti-cancer therapeutics, and by better understanding the inner workings of the motors, it is hope that this work will contribute to these efforts.
- Dissertation Note:
- M.S. Pennsylvania State University 2017.
- Technical Details:
- The full text of the dissertation is available as an Adobe Acrobat .pdf file ; Adobe Acrobat Reader required to view the file.
View MARC record | catkey: 20513770