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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - ACPNMR (Structural dynamics of acyl carrier protein complexes through combined solution and solid-state NMR)

Teaser

Summary

The aim of the proposed project was to obtain molecular level understanding of protein interactions in complexes from biosynthetic factories for natural products active against bacterial pathogens. Natural products including polyketides and non-ribosomal peptides comprise 20% of the top-selling drugs, with combined worldwide annual revenues of over Â£10 billion. Due their complex structures, they are often difficult to synthesize by chemical methods. However, a synthetic biology approach where the natural products are produced biosynthetically by genetically manipulated assembly lines, which include non-ribosomal peptide synthases (NRPSs) and polyketide syntheses (PKSs), is becoming increasingly popular. Inactivation, substitution or addition of new domains to the naturally occurring modules, enables the biosynthesis of compounds that are not found in nature and introduce desirable properties into natural products. However, engineering of NRPSs/PKSs often leads to assembly lines that are less efficient than the original multienzymes or sometimes not active. Knowledge of the structural dynamics exhibited by the NRPSs is therefore essential to enable their engineering, especially if such strategies are to be ever implemented in an industrial scale production. As a general approach we proposed to use a combined solution and solid-state NMR to study such systems.
In particular, in this project we have investigated systems involved in important biosynthetic step of halogenation (specifically attachment of chlorine). Using multidisciplinary approach including biochemical assays, biophysical methods, NMR, mass spectrometry and X-ray crystallography we obtained initial insight for the mechanism controlling the concerned reaction.

Work performed

\"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 Î²-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 Î²-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 Î²-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 Î²-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 Alzheimerâ€™s Î²-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.\"

Final results

Our research projects covered by Marie SkÅ‚odowska-Curie proposal have initiated studies in intriguing scientific areas. We have demonstrated specific interaction of curcumin with Î²-amyloid oligomers, which opens new field for further interesting studies. Our project concerning proteins of aeruginosin/cyanopeptolin proteins, have reached the stage where we know how to prepare stable solution NMR samples, we recorded good quality 13C, 15N TOCSY and NOESY spectra, and obtained crystals of one of the most interesting in our project protein - halogenase. The preliminary data on characterisation of interactions and function of proteins from aeruginosin system has provided basis for a successful BBSRC grant application on synthetic biology of these systems, which will provide the next step for translating the biophysical data into rational engineering of biosynthetic pathways.
At Warwick, I was part of a highly stimulating scientific environment of scientists from all around the world, giving me the unique opportunity to discuss, exchange ideas, and learn new points of view. Moreover, the possibility to have an access to a variety of high end scientific equipment allowed me to perform experiments that I had never had an opportunity to perform before. The possibility to share my experience with Lewandowski group members and to get their rather unique knowledge in biomolecular solid-state and solution NMR was undoubtedly a great opportunity to gain scientific maturity and independence, which are cornerstones for my future career. Now I am able to establish my own research group, become an independent scientist and obtain a habilitation within a short amount of time (I have already full-time position at the University of Wroclaw).
I took part in transferable skills workshops, e.g. concerning open access publishing, proper data management or career planning offered by Warwick Learning and Development Centre. I also attended Marie SkÅ‚odowska-Curie workshop (School of Life Sciences) where I was invited as a speaker to present successful approach while writing MSC proposal.
I took part in Warwick School Outreach program (St Margaret\'s CofE Junior School, Lamington), where I assisted my colleague Marianne Costa. Children, age 10 and 11 together with their parent were introduced to the difficult problem of scientists battle with pathogenic bacteria in an appropriate, accessible way.