Structural investigations of microtubule-associated motor proteins by magic angle spinning solid-state NMR spectroscopy.

摘要

Despite the importance of the assemblies formed by microtubules and microtubule-binding proteins (MTBPs), there is no structural or dynamics information about these assemblies at the atomic level because they are very large, insoluble and lack long-range order. These properties make them elusive targets for the two most prevalent structural biology techniques, X-ray crystallography and solution NMR spectroscopy.;Magic angle spinning (MAS) solid-state NMR spectroscopy has recently emerged as a promising technique for structural analysis of biomacromolecules, and its unique abilities to provide atomic-level structural and dynamics information in insoluble noncrystalline systems coupled with no intrinsic size limitation make it particularly suitable for studies of large proteins and protein assemblies. In the past five years, our group has been working on methodological developments to enable MAS solid-state NMR structural investigations of MT/MTBP assemblies with the long term goal of applying this technique to a broader range of cytoskeleton-related biological systems.;This dissertation concerns i) structural studies of CAP-Gly domain free and in complex with microtubules, ii) structural studies of dynein light chain LC8, and iii) methodological MAS NMR developments aimed at sensitivity and resolution enhancements, which enable studies of microtubule-associated protein assemblies. We discuss resonance assignments and secondary structure analysis of CAP-Gly and LC8, by multidimensional MAS NMR and solution NMR spectroscopy. We present the first high-resolution MAS NMR spectra for microtubule-bound CAP-Gly, and discuss the observed multiple chemical shift perturbations, which indicate conformational changes of CAP-Gly upon binding to microtubules. These results validate the feasibility of using MAS solid-state NMR spectroscopy for structural investigations of MT/MTBP assemblies. We report a novel sensitivity and resolution enhancement MAS NMR method developed by us and dubbed NUS-PACC that can use less than 4 percent of the experiment time to achieve the same sensitivity and resolution offered by conventional experimental approaches. This development enables investigations of sensitivity-limited samples, in particular, MT/MTBP assemblies. The studies detailed in this dissertation established the groundwork for successful accomplishment of our long-term objectives.