Structural and dynamics studies of microtubule-associated Tau and Kif5b assembled with microtubules; NMR crystallography of oxovanadium (V) bioinorganics: insights from magic angle spinning NMR spectroscopy

Li, Mingyue
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University of Delaware
Magic angle spinning (MAS) NMR is an important technique in the structural and dynamics characterization of biomacromolecules and bioinorganic complexes, especially those which lack solubility or resist crystallization. The knowledge of structure and dynamics of biological systems is crucial to understanding their functions. As systems of interest become increasingly challenging regarding the molecular size and complexity, novel MAS NMR strategies for characterization of proteins and bioinorganic solids are developing rapidly. This thesis discusses the application of MAS NMR spectroscopy to study structure, dynamics, and chemical/biological functions in two classes of systems. The first is microtubules (MTs) and microtubule-associated proteins (MAPs); the second is vanadium(V)- containing bioinorganic solids. ☐ The MAP Tau plays critical roles in regulating multiple MT activities in neurons through binding with MTs. The loss of Tau binding to microtubules is pathologically relevant to many neurodegenerative diseases including Alzheimer’s disease (AD). I present an investigation into the conformation and dynamics of Tau assembled with polymerized MTs by MAS NMR spectroscopy. Our studies of TauF4, a functional fragment of Tau, demonstrate that, surprisingly, TauF4 remains disordered upon binding with MTs and is highly dynamic on a wide of range of timescales (nano- to milliseconds). To my knowledge, such remarkable mobility of a protein when bound to its partner is unique to TauF4 and is consistent with its function in regulation of MT polymerization dynamics. ☐ The Microtubule-associated motor protein Kif5b is essential to intracellular transport and cell division. Determining Kif5b motor domain structure assembled with polymeric microtubules is crucial for understanding the mechanism of Kif5b translocation along MTs. The protocol for preparing Kif5b/MT assemblies as presented in the thesis is the first for MAS NMR studies. Our preliminary NMR studies of Kif5b/MTs demonstrate that Kif5b bound to microtubules is well-structured, and lay the methodological foundation for its 3D structure determination. ☐ Vanadium bioinorganic solids have found diverse applications as catalysts and demonstrated potential utility as pharmaceuticals. Vanadium in its +5 oxidation state is diamagnetic and not amenable to conventional EPR and UV-vis characterizations but is well suited for solid-state NMR studies. In deriving the structures of vanadium (V)-containing bioinorganic compounds, we combine multinuclear NMR spectroscopy with density functional theory (DFT) calculations. This approach, termed NMR crystallography, is first validated in a structurally known compound (15N-salicylideneglycinate)-(benzhydroxamate)oxovanadium(V) (VO(15NGlySal)bz) and subsequently employed in the determination of the three-dimensional structure of (methoxo)(15N-salicylidene-glycinanto)oxovanadium(V) (abbreviated as VO(15NGlySal)OCH3(CH3OH)) including the correct solvation state, which has not been reported previously. This work has expanded the application of NMR crystallography in the context of vanadium(V)-containing compounds.
Pure sciences , Alzheimer's disease , Crystallography , Motor protein , NMR , Protein dynamics , Protein structure