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High Resolution Two-Dimensional Detection of Ncd Motility with Optical Tweezers

Published online by Cambridge University Press:  15 February 2011

M. W. Allersma ,
F. Gittes ,
M. J. deCastro ,
R. J. Stewart  and
C. F. Schmidt
M. W. Allersma
Affiliation:
University of Michigan, Dept. of Physics & Biophys. Res. Div., Ann Arbor, MI 48109
F. Gittes
Affiliation:
University of Michigan, Dept. of Physics & Biophys. Res. Div., Ann Arbor, MI 48109
M. J. deCastro
Affiliation:
University of Utah, Dept. of Bioengineering, Salt Lake City, Utah 84105.
R. J. Stewart
Affiliation:
University of Utah, Dept. of Bioengineering, Salt Lake City, Utah 84105.
C. F. Schmidt
Affiliation:
University of Michigan, Dept. of Physics & Biophys. Res. Div., Ann Arbor, MI 48109
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Abstract

The ATP-dependent motility of the kinesin-related non claret disjunctional (ncd) mechanoenzyme was observed in an in vitro bead motility assay using optical tweezers in combination with a new two-dimensional displacement detection method. The detection technique is based on observing the far-field interference pattern formed in the back focal plane (BFP) of the microscope condenser by the illuminating laser focus and the light scattered from the trapped dielectric bead. The ability to observe the two-dimensional motion, with high temporal and spatial resolution, and in a manner largely independent of position in the microscope field-of-view, is the particular advantage of this detection method. In the assay, a fusion protein (GST-N195) of truncated ncd and glutathione-Stransferase was adsorbed to silica beads and the axial and lateral motions of the beads along the microtubule surface were observed. The average axial velocity of the ncd coated beads was 230 ± 30 nm/s (± std. dev.). Spectral analysis of bead motion showed an increase in viscous drag near the surface. Furthermore, we also found that any elastic constraints of the moving motors are much smaller than the constraints due to binding in the presence of the non-hydrolyzable nucleotide adenylylimido-diphosphate (AMP-PNP).

Type
Research Article
Information
MRS Online Proceedings Library (OPL) , Volume 489: Symposium K – Materials Science of the Cell , 1997 , 85
Copyright
Copyright © Materials Research Society 1998

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