Dynamic Studies Of Abscsic Acid(ABA)Receptor PYL10Using Solution Nuclear Magnetic Resonance Upon ABA Binding

Protein is one of the most important biomacromolecules for biological functions. Most of protein function is predicated on dynamics, and better understanding on significance of life can be achieved by mechanism studies of protein in physiological processes.. NMR spectroscopy is a power tool for studying protein dynamic and mechanism since a wide range of time scales. PYL10is one of the member of Arabidopsis thaliana soluble abscisic acid(ABA) receptor family (RCAR1/PYR/PYL) inhibiting its downstream signal factor, type2C protein phosphatase(PP2C). In this thesis, we employ some biological experimental methods together with NMR to investigate biological functionof PYL10. A conclusion that ABA plays an important role in the PP2C inhibiting function by PYLO, rather than "PYL10is an ABA-independent receptor" revealed by previous study, is generated according to our data. The dynamic assay of PYL10upon ABA binding was further investigated using liquid NMR relaxation and reduced spectral density function methods. The result shows ABA binding brings an increase of comformational entropy and protein flexibility of PYL10. Meanwhile, some other work which using mass spectrometry-based label-free quantitative method to perform site-specific quantitative analysis of Tyr574auto-phosphorylation of the Escherichia coli tyrosine kinase C-terminal catalytic domain(ETK-CTD) is also introduced in this thesis.In chapter one a brief introduction on the finding and classification of ABA receptor is given at the beginning. And then we describe the main content and function of the ABA signal pathway mediated by soluble RCAR1/PYR/PYL ABA receptor family. At the end of chapter one, we describe the structural changes in ABA perception and the mechanism of RCAR1/PYR/PYL family inhibiting phosphatase activity of PP2C.Chapter two is a brief review of methods on protein dynamic assay based on liquid NMR spin relaxation. Studies have shown that the combination of thermodynamics and dynamic can explain the protein-ligand or protein-protein integration mechanism reasonable.In chapter three, the chromogenic reaction of molybdate and phosphate was taken to investigate osmotic pressure influence on ABA receptor PYL10and PYL2inhibiting the PP2C(HAB1) phosphatase activity. The result shows PYL10inhibits HAB1phosphatase activity is ABA-dependent and would be influenced by BSA which is contrary to previously ABA-dependent conclusion. Further biochemical assay shows that PYL10and BSA wouldn’t form a stable complex.Based on the conclusion that BSA doesn’t influence the function of PYL10via direct interaction, a corollary that this effect may come from the dynamic changes of PYL10. To investigate this, in chapter four, a lot of experiments were taken including the backbone assignment, relaxation data collection and reduced spectral density function assay of free state PYL10(apo-PYL10) and ABA/PYL10complex and the titration assay, a conclusion that the increasing inhibition of PP2C activity by PYL10is the result of PYL10conformation entropy and flexibility increase during ABA binding was achieved.In chapter five, we used the label-free mass relative quantification assay and western-blot qualitative assay to investigate the phosphorylation level changes of ETK-CTD autophosphorylation site Tyr574and the total protein phosphorylation level respectively. The mass spectrum result shows that the phosphorylation level of ETK-CTD Tyr574reaches a low but stable rate(about1%) very quickly. However, the western-blot assay indicates the total protein phosphorylation level remains elevated eventhough Tyr574phosphorylation reaches the stable rate. These data strongly suggest that a low level of auto-phosphorylation is sufficient to trigger cross-phosphorylation to reach a steady state of total phosphorylation.The fourth chapter is a brief introduction of some preparatory work on plant inward rectifier potassium channels KAT1.