Brownian Dynamics Simulation of Kinesin

Open Access
Author:
Cook, Zeke
Area of Honors:
Biomedical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • William O Hancock, Thesis Supervisor
  • William O Hancock, Honors Advisor
  • Justin Lee Brown, Faculty Reader
Keywords:
  • Kinesin
  • Protein
  • Motor
  • Mechanobiology
  • Mechanochemistry
  • Processive
  • Fluorescence
  • Gold Nanoparticle
  • Tracking
  • Total Internal Reflection
  • Microscopy
  • Interferometric Scattering
  • Brownian
  • Dynamics
  • Brownian Dynamics
  • Molecular Modeling
  • Molecular Dynamics
Abstract:
High frequency nanoparticle tracking has emerged in recent years as a powerful strategy to track the nanoscale dynamics of proteins, DNA, and other biomolecules. Using dark-field or interferometric scattering microscopy techniques (iScat) in conjunction with point-spread fitting, sub-millisecond temporal resolution images of nanoparticle-labeled proteins can be achieved with nanometer spatial precision. However, there remain unresolved questions regarding the best practices for site-specific tagging of proteins. In this thesis, Brownian dynamics simulations and artificial image generation techniques are employed to investigate the influence of tether length, tether stiffness, and particle size on imaging precision and the ability to accurately measure protein fluctuation dynamics. The first finding is that point spread function fitting cannot be extended to arbitrarily fast frame rates because the gold probe undergoes tethered diffusion about its anchor point. One implication is that camera exposure times that allow the probe to fully explore its positional distribution increase fitting precision. The second finding is that changes in protein rotational freedom give rise to artifactual translations of the nanoparticle position that do not necessarily correlate with actual protein dynamics. These lessons are employed to interpret experimental data of gold nanoparticle-labeled kinesin motor proteins stepping along microtubules. It is found that apparent discrepancies in the literature regarding the structure of the kinesin dimer in the ATP waiting state are due to the differences in label strategy and gold attachment position. These results emphasize the importance of carefully considering controllable conjugation and imaging parameters when designing and interpreting gold nanoparticle tracking experiments and provide new insights into the protein dynamics underlying the kinesin mechanochemical cycle.