"The project covered three main topics: 1) initial target: gladiolin polyketide synthase (PKSs) subunits interaction (acyl carrier protein - ACP, ketosynthases - KS), 2) aeruginosin/cyanopeptolin nonribosomal polyketide synthases (NRPS) subunits interaction (peptydyl carrier protein - PCP, halogenase - H and condensation domain - C), 3) interaction of curcumin with β-amyloid oligomers. The initial target for the studies, ACP from gladiolin PKS system was unstable in NMR buffers so sufficiently long times, so we decided to change the consider alternative but related systems from aeruginosin/cyanopeptolin NRPS. Expression and optimisation of conditions for production of gladiolin ACP took us around 5 months. From aeruginosin/cyanopeptolin systems, we have expressed biosynthesised and optimized conditions for six different proteins (2 x PCP, 2 x H and 2 x C). For aeruginosin halogenase we obtained crystals and synthetized substrate for activity assay - publication is planned in the middle of 2018. Additionaly, for two PCPs, we have collected 13C and 15N 3D solution NMR spectra and we are on a good way to obtain the structures.
As a training and preparation for applying solid-state NMR to the complexes from aeruginosin NRPS we have investigated interactions between curcumin and β-amyloid oligomers. In this subproject, we established presence of specific interaction between curcumin and oligomers by applying new type of cross-polarisation based 2D correlation spectra, where natural abundance oligomers were used. These studies demonstrating specific interaction with β-amyloid oligomers provide basis for search for oligomer binders that could lead to therapeutics in the future. The project was performed in the collaboration with Prof. Oleg Antzutkin from Lulea University of Technology, Sweden whose group have prepared β-amyloid oligomers and specifically 13C labelled cuyrcumin. All NMR experiments were performed in the NMR facility at Warwick University. The publication from this work is in preparation.
Exploitation and dissemination of the results include mostly my attendence in interational conferences and workshops. In 2016, I attended UK 850 MHz solid-state NMR facility annual symposium and The Chemistry and Biology of Natural Products Symposium XI. In 2017, to disseminate the results, I participated in two international conferences, where I presented posters: 1) Directing Biosynthesis Conference V (Royal Society of Chemistry), ""Investigating the interaction between halogenases and peptidyl carrier proteins involved in aeruginosin and cyanopeptolin biosynthesis"", Warwick, UK and 2) ENC conference - 58th Experimental Nuclear Magnetic Resonance Conference, ""Insights into the Specific Interaction of Alzheimer’s β-amyloid Toxic Oligomers with 13C Carbonyl-labelled Curcumin"", Pacific Grove, California. One scientific paper is in preparation, second is planned in the middle of 2018."
