Insights into Kinesin-1 Stepping from Simulations and Tracking of Gold Nanoparticle-Labeled Motors

Abstract

High-resolution tracking of gold nanoparticle-labeled proteins has emerged as a powerful technique for measuring the structural kinetics of processive enzymes and other biomacromolecules. These techniques use point spread function (PSF) fitting methods borrowed from single-molecule fluorescence imaging to determine molecular positions below the diffraction limit. However, compared to fluorescence, gold nanoparticle tracking experiments are performed at significantly higher frame rates and utilize much larger probes. In the current work, we use Brownian dynamics simulations of nanoparticle-labeled proteins to investigate the regimes in which the fundamental assumptions of PSF fitting hold and where they begin to break down. We find that because gold nanoparticles undergo tethered diffusion around their anchor point, PSF fitting cannot be extended to arbitrarily fast frame rates. Instead, camera exposure times that allow the nanoparticle to fully populate its stationary positional distribution achieve a spatial averaging that increases fitting precision. We furthermore find that changes in the rotational freedom of the tagged protein can lead to artifactual translations in the fitted particle position. Finally, we apply these lessons to dissect a standing controversy in the kinesin field over the structure of a dimer in the ATP waiting state. Combining new experiments with simulations, we determine that the rear kinesin head in the ATP waiting state is unbound but not displaced from its previous microtubule binding site and that apparent differences in separately published reports were simply due to differences in the gold nanoparticle attachment position. Our results highlight the importance of gold conjugation decisions and imaging parameters to high-resolution tracking results and will serve as a useful guide for the design of future gold nanoparticle tracking experiments.

A kinetic dissection of the fast and superprocessive kinesin-3 KIF1A reveals a predominant one-head-bound state during its chemomechanical cycle

“…1), it has been established that, following initial binding and release of ADP (state 3), kinesin-1 waits for ATP binding with the tethered head in a rearward position. 27,32,33 ATP binding to the bound head then repositions the tethered head forward, and ATP hydrolysis triggers full neck linker docking, which positions the tethered head near its next binding site. The forward step is completed by the tethered head binding the microtubule and releasing its bound ADP to generate a tightbinding state 7.…”

Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end

“…Tapered microtubule plus-ends were designed as a uniform distribution of protofilament lengths with stepwise increases in length occurring linearly from the 1 st and 13 th protofilament, making the center protofilament the longest. The average photon emission per tubulin subunit was determined by measuring the magnitude of the microtubule signal (1160 arbitrary intensity units, corresponding to a 5.2% contrast signal) relative to the average signal from back reflected light (22300 units) on a 16-bit image (65535 units maximum) (Mickolajczyk et al, 2019a). A mask was defined and the microtubule placed randomly along a 1024 x 1024 pixel image (59.4 x 59.4 μm).…”

Long-range intramolecular allostery and regulation in the dynein-like AAA protein Mdn1

“…where γ = 6πηr is the drag coefficient of a sphere with radius r in fluid with viscosity η (set equal to the viscosity of water), D is the diffusion constant, and F therm is a Gaussian white noise process with mean zero (55). This equation was integrated numerically using modified Euler's method, such that x(t) was updated every 1-ns time step (Δt) by:…”