Pseudomonas sp. JHN
National Institutes of Health
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.................................................................................................................................... ii Table of contents ...................................................................................................................... iv List of figures ........................................................................................................................... vi List of tables ............................................................................................................................ vii Acknowledgements ................................................................................................................... x Chapter 1: Introduction ............................................................................................................. 1 1.1 Post-translational modifications ................................................................................................... 1 1.2 O-GlcNAcylation defines a new class of glycoproteins ............................................................... 1 1.3 GlcNAcylation enzymes: OGT and OGA .................................................................................... 4 1.4 Crosstalk between O-GlcNAcylation and phosphorylation ......................................................... 7 1.5 O-GlcNAc and Alzheimer’s disease related protein tau............................................................... 8 1.7 Thesis overview .......................................................................................................................... 12 Chapter 2: Materials and methods .......................................................................................... 15 2.1 Cloning ....................................................................................................................................... 15 2.1.1 His6-SUMO-tau 353-408 ........................................................................................................... 15 2.1.2 Site-directed mutagenesis for aspartic acid mutants ............................................................ 15 2.2 Protein expression and purification ............................................................................................ 17 2.3 NMR spectroscopy ..................................................................................................................... 19 2.4 Aggregation assays ..................................................................................................................... 20 Chapter 3: NMR spectral assignments .................................................................................... 22 3.1 Assignments from main-chain nuclei ......................................................................................... 22 3.2 Cis/trans conformational isomers of tau .................................................................................... 27 3.3 Assignment of O-GlcNAc NMR signals .................................................................................... 28 Chapter 4: Structural, functional, and dynamic studies of tau ................................................ 31 4.1 Chemical shift perturbations ....................................................................................................... 31 4.2 Secondary structure propensities derived from chemical shifts ................................................. 31 4.2.1 δ2D ...................................................................................................................................... 31 4.2.2 3 JHN-Hα coupling .................................................................................................................... 32 4.2.3 NOESY experiments ........................................................................................................... 33 4.3 15 N Relaxation ............................................................................................................................. 37 4.4 Cis/trans isomerization ............................................................................................................... 39 4.5 Aggregation assays ..................................................................................................................... 40
Huntingtonova nemoc (HN) je autozomalně dominantně dědicne neurodegenerativni onemocněni s výskytem cca 1 : 10–15 000. Podstatou…