AbstractThe plant cell cycle is a crucial mechanism in plant development. The main components for the regulation of this cycle are cyclins (CYC) and cyclin-dependent kinases (CDK). These two groups of proteins can form complexes that allow the cell to progress in the cell cycle by phosphorylating certain other proteins. However, in order to adapt to environmental changes, organisms need extra regulation in the form of CDK inhibitors (CKI). These CKIs are able to bind to specific CDK-CYC complexes and inhibit their activity.
This thesis aimed to help understand the plant cell cycle, since less is known about this mechanism than the cell cycle of yeast or animals. Until now, no plant CDK-CYC complex structures have been solved, while there are structures of the yeast and animal homologs. For this purpose, CDKA;1 and CYCD2;1 from Arabidopsis thaliana were used with the inhibitors KRP2 and SMR7.
It was possible to express the CDKA;1-CYCD2;1 complex in insect cells using the Bac-to-Bac system and purify the complex with immobilized metal ion affinity chromatography and gel filtration. The purified complex was used in an activity assay where the inhibiting activity of several peptides was tested. A peptide derived from KRP2 was able to inhibit the CDKA;1-CYCD2;1 complex, while no inhibiting activity was observed from the three peptides derived from SMR7. The KRP2 peptide was then used in an isothermal titration calorimetry experiment with the CDKA;1-CYCD2;1 complex to analyze the binding characteristics of the peptide to the complex.
The results from the isothermal titration calorimetry were compared with p27. Indications that KRP2c is an intrinsically disordered protein that folds upon binding, were found.
|Date of Award||29 Jun 2015|
|Supervisor||Remy Loris (Promotor), Ariel Talavera Perez (Advisor), Serge Muyldermans (Jury) & Gustavo Gutierrez Gonzalez (Jury)|
- Cell cycle
- Cyclin-dependent kinase
- protein chemistry
- Plant cell cycle
- Intrinsically disordered proteins
- Structural Biology