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
Released Data Link
Team
Principal Investigator
Team Members
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