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High-Resolution Magic Angle Spinning NMR Spectroscopy of Microtubule-Associated Motor Proteins


EMSL Project ID
26700

Abstract

This is an application in response to the capability-based call "Bio-Solids Capability on the 900 MHz NMR". We request standard, general access to the 900 MHz solid-state NMR instrument at EMSL. Two- and three-dimensional homo- (C-C) and heteronuclear C-N) correlation magic angle spinning NMR spectra of microtubule-associated motor proteins of human dynein and dynactin will be acquired. Dynein and dynactin proteins form a large superfamily of motor proteins responsible for a number of intracellular transport processes along the microtubules. Dynein and dynactin proteins have been implicated in pathogenesis of multiple disorders, such as motor neuron diseases and degenerative disease; up-regulation of dynein light chain 1 has been linked to promoting tumorigenesis. The objective of our study is to gain understanding of the structure and dynamics of the DLC8 protein and of the CAP-Gly domain of the p150Glued subunit of dynactin in the free form and bound to the microtubules. The microtubule assembly of CAP-Gly is a high molecular weight complex not amenable to X-ray crystallography or solution NMR spectroscopy, and we will employ state-of-the-art high-field solid-state MAS NMR to obtain high-resolution multi-dimensional spectra of the uniformly 13C,15N- enriched CAP-Gly preparation that will be subsequently analyzed to gain structural and dynamc information. These experiments will complement the measurements at 14.1 T conducted in our laboratory. The use of the 21.1 T spectrometer is necessary for this work as it will allow us to gain the resolution and sensitivity necessary for characterization of the uniformly enriched microtubule-bound complex of CAP-Gly.

Project Details

Project type
Capability Research
Start Date
2007-10-01
End Date
2009-07-27
Status
Closed

Team

Principal Investigator

Tatyana Polenova
Institution
University of Delaware

Team Members

Yun Han
Institution
University of Delaware

Sivakumar Paramasivam
Institution
University of Delaware

Shangjin Sun
Institution
University of Delaware

Related Publications

Lightcap CM, S Sun, JD Lear, U Rodeck, TE Polenova, and JC Williams. 2009. "Biochemical and Structural Characterization of the Pak1-LC8 Interaction." Journal of Biological Chemistry 283(40):27314-27324. doi:10.1074/jbc.M800758200
Sun S, AH Butterworth, S Paramasivam, S Yan, CM Lightcap, JC Williams, and TE Polenova. 2011. "Resonance Assignments and Secondary Structure Analysis of Dynein Light Chain 8 by Magic-angle Spinning NMR Spectroscopy." Canadian Journal of Chemistry 89:909-918. doi:10.1139/V11-030
Sun S, A Siglin, JC Williams, and TE Polenova. 2009. "Solid-State and Solution NMR Studies of the CAP-Gly Domain of Mammalian Dynactin and Its Interaction with Microtubules." Journal of the American Chemical Society 131(29):10113-10126. doi:10.1021/ja902003u