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Biophysical characterisation of a protein-activating protein-protein interaction

Periodic Reporting for period 1 - Protprot (Biophysical characterisation of a protein-activating protein-protein interaction)

Reporting period: 2015-08-01 to 2017-07-31

"Plants have found numerous strategies to adapt to a changing climate. On such strategy, C4 photosynthesis, is critical to how crop plants such as maize and sorghum can thrive in hot and dry climates. While we understand the chemistry of this adaptation, we do not understand the biochemistry and how precisely it is regulated and evolved. Deepening our klnowledge about the ""engines"" of C4 type photosynthesis is valuable for biotechnology and can have applications in improving the yields of other plants used as human food.
In this project, we aimed to use biophysical approaches to understand the determinants of interaction between a protein central to the C4 pathway for photosynthetic CO2 fixation (PPDK) and the regulatory kinase with which it interacts. We wanted to elucidate the molecular mechanism of that unusual kinase that is dependent upon ADP rather than ATP. Our studies on that regulatory protein (PDRP) will also aid our understanding of other kinase enzymes.
In particular, we aimed to use recently-described methodology for the incorporation of phosphoserine into proteins to generate per-phosphorylated proteins for model binding studies as well as chemical approaches developed in the host laboratory for the generation of model peptides containing analogues of phosphohistidine. That is because the interaction between PPDK and PDRP proteins is dependent upon the presence of a phosphothreonine residue in PPDK. It was shown that PPDK with phosphoserine in the place of phosphotreonine remains a substrate for PDRP. We also aimed to investigate the interaction of the regulatory protein with its small molecule ligands.
The final project took part in two phases. Since the proposed project was chiefly designed to enable Dr Witkowska to obtain training in protein purification and characterisation, we initially worked on a simpler alternative model protein ATIC to characterise ligand binding in this system. A report of this work has now been submitted for publication is under consideration having been revised following peer review. The methods and techniques learnt with this simpler system (in which a protein is interacting with a commercial ligand rather than a protein with a protein modified in a complex manner) were then transferred to the main target system of the project.
Following optimisation of protein expression and purification, the maize PDRP was submitted for crystallisation trials and small molecule binding characterised by ITC to determine the molecular basis for the ADP-specificity of this kinase. We investigated the effect of truncation of the stability of the target protein in light of a recently reported crystal structure. The results of these studies are currently being prepared for publication.
The phosphorylated target protein (PPDK) was generated using both chemical and genetic incorporation methods and its interaction with PDRP assessed using ITC. Based on these initial studies, we are continuing the project using alternative protein substrate expression constructs. Results of this work have already been shown internationally and nationally at conferences (e.g. FEBS Congress in Jerusalem or Chemistry Postdoctoral Conference in Leeds).
"The project focused on aspects of protein-protein interaction analysis and allowed the fellow (Dr Witkowska) to work in a multidisciplinary environment between chemistry and biochemistry. During the project, the Dr Witkowska, was trained and gained expertise in key techniques for protein overexpression and purification (SDS-page, SEC), in biophysical characterisation of these materials (ITC, SPR) and in advanced techniques for protein chemistry such as the inclusion of unnatural amino acids and labeling of proteins.
This fellowship enabled D.Witkowska to receive permanent work in her country after coming back from UK (she has been working at the Opole medical school since the 1st of August 2017). She also has applied for the grant to Foundation for Polish Science, wishing to continue her work on protein-protein interactions, and is waiting for the assesment of her proposal. Throughout her studies at Leeds Unversity Dr Witkowska had the opportunity to supervise a few of junior researchers. The ""proprot"" project enabled her to disseminate the new knowledge to her students and coworkers and also to set new collaboration in Leeds and Durham which will help her to set up her own lab and put forward the science in her home country.
Reaction mechanism of PPDK and PDRP studied in this project