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Kinesin / Microtubule Molecular Motor System
Effects of casein surface passivation in kinesin-1 gliding motility assay
Maloney A, Herskowitz LJ, Koch SJ (2011) Effects of Surface Passivation on Gliding Motility Assays. PLoS ONE 6(6): e19522. http://dx.doi.org/10.1371/journal.pone.0019522
Andy Maloney studies the effects of alpha, beta, kappa, and whole casein in the kinesin-1 gliding motility assay. Automated speed analysis software by Larry Herskowitz.
Figure: Black circles are alpha casein passivation, red squares are beta casein passivation, green up pointing triangles are whole casein passivation, and blue down pointing triangles are mixed casein passivation. Each data point is the mean from three different samples, taken at approximately the same assay time. Error bars represent the standard error of the mean. Alpha casein had the most consistent average speed measurements at 949±4 nm/s. Whole casein and mixed casein averaged to 966±7 nm/s and 966±6 nm/s respectively. Beta casein averaged to 870±30 nm/s.
A discrete-state model for kinesin-1 with rate constants modulated by neck linker tension (under review, PLoS ONE)
Koch, Steven and Herskowitz, Lawrence. A Discrete State Model for Kinesin-1 with Rate Constants Modulated by Neck Linker Tension. Available from Nature Precedings http://hdl.handle.net/10101/npre.2010.5038.1 (2010)
In George Bachand's lab with Susan Rivera, fungal kinesin from Thermomyces lanuginosus
Rivera SB, Koch SJ, Bauer JM, Edwards JM, Bachand GD. 2007. "Temperature dependent properties of a kinesin-3 motor protein from Thermomyces lanuginosus." Fungal Genetics and Biology 44:1170-1179. PMID 17398126
Figure: Dynamic light scattering data showing the inhibition by ATP of heat-induced aggregation of kinesin from Thermomyces lanuginosus.
More on kinesin aggregation
Probing Protein-DNA Interactions by Unzipping DNA with Optical Tweezers
Overview of the shotgun DNA mapping method. We'll have to get rid of the beer can for the final publication.
Proof of principle for shotgun DNA mapping by unzipping
Our initial proof-of-principle publication in the Wang Lab at Cornell
Figure 2A from the Biophys. J. paper. The black trace shows unzipping force for a single DNA molecule in the absence of protein. The red trace shows in the presence of a DNA-binding protein, with predicted binding curves shown as dotted lines. Unoccupied binding sites marked with arrows.
Koch SJ, Shundrovsky A, Jantzen BC, Wang MD. Probing protein-DNA interactions by unzipping a single DNA double helix. Biophys J. 2002 Aug;83(2):1098-105. PMID 12124289
Our follow-on paper showing that unbinding forces can be analyzed nicely with Evan Evans' Dynamic Force Spectrosocpy (DFS) model
Fig. 3 from the PRL. Orange bars are histograms of 449 total unbinding events. Dashed curves are maximum likelihood fits of the PDF from Evan Evans' DFS model, each fit to a single rate. Solid lines are PDFs from a single best fit for all rates. Vertical dashed bars represent unaccessible ranges to our experiment.
Koch SJ, Wang MD. Dynamic force spectroscopy of protein-DNA interactions by unzipping DNA. Phys Rev Lett. 2003 Jul 11;91(2):028103. PMID 12906513
- Buried in this paper is the "loading rate clamp" that we used and which greatly simplifies data analysis as well as provides much cleaner data. Also, our maximum likelihood method for data analysis is better than the typical method of fitting Gaussians to histograms, but this was also buried in footnotes. It's been while since published, but the Koch lab would like to publish the details of these methods, as they would be very helpful to others doing DFS.
DNA in porous nanochannels
Initial passive transport of DNA in porous nanochannels fabricated from silica nanoparticles
The first two panels are SEM of nanochannels with silica nanoparticle walls. The third (right) panel is a confocal fluorescence image of lambda DNA accumulated in porous nanochannels.
We have been consulting with the Brueck and Lopez labs at UNM on a project for transporting DNA in nanochannels where the walls are formed from silica nanoparticles are are thus porous on the nanoscale. Initial work of Deying Xia and Thomas Gamble were recently published in Nanoletters:
Xia Deying, Gamble Thomas C., Mendoza Edgar A., Koch Steven J., He Xiang, Lopez Gabriel P., and Brueck S. R. J. "DNA Transport in Hierarchically-Structured Colloidal-Nanoparticle Porous-Wall Nanochannels." Nano Lett., 8 (6) 1610 - 1618, 2008
MEMS Force Sensor for Biophysics
Work done with Gayle Thayer, Alex Corwin, Maarten de Boer at Sandia, finished up after Koch moved to UNM, Physics & CHTM
Scanning Electron Microscope image of a force sensor similar that used for the research.
Koch SJ, Thayer GE, Corwin AD, de Boer MP. Micromachined piconewton force sensor for biophysics investigations. Appl. Phys. Let. 2006 Oct 23;89(17):173901 (PDF)