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Non-ribosomal peptide synthetases (NRPSs) are large enzymatic systems responsible for the biosynthesis of a wealth of secondary metabolites, many of which are used by pharmaceutical scientists to produce drugs such as antibiotics or anticancer agents. To synthesize all of these remarkably diverse compounds, bacteria and fungi use a surprisingly conserved strategy: NRPSs are organized in modules, made of conserved domains, that each incorporates a dedicated substrate. Thus, new compounds with improved activities can be generated, in principle, by swapping domains or modules in order to control substrate incorporation and hence the final product. To do this efficiently, a deep understanding of domain communication during the synthesis is required. NMR and crystallography studies indicate that domain interactions are transient and that their quaternary structure is likely subject to rearrangements during the synthesis. In addition, both techniques show that individual domains are subject to conformational heterogeneity. We principally use NMR to investigate inter- and intra-domain modifications occurring during the catalytic steps of non-ribosomal peptide synthesis.
Development of NMR methods. The enzymatic systems that we investigate provide many challenges to NMR. Some of the domains or multi-domains are large, and therefore, the resulting data suffer from spectral crowding and signal losses. In addition, the proteins are subject to dynamics, which may further deteriorate the quality of the spectra.Consequently, new methods often need to be developed to overcome these challenges. The techniques provide means to assign the NMR signals to the atoms in the proteins, to measure structural constraints, or to monitor dynamics. When faced with an impasse, new techniques can be designed in our lab. With these new techniques available, new protein targets can be considered, thereby increasing our overall understanding of the systems. Thus, we foster a synergy between studies of relevant biological systems and the development of new NMR techniques.
Harden BJ, Mishra SH, Frueh DP. J Magn Reson. Effortless assignment with 4D covariance sequential correlation maps. 2015 Nov;260:83-8. doi: 10.1016/j.jmr.2015.09.007. Epub 2015 Sep 21.PMID:26432397
Goodrich AC, Harden BJ, Frueh DP. Solution Structure of a Nonribosomal Peptide Synthetase Carrier Protein Loaded with Its Substrate Reveals Transient, Well-Defined Contacts. J Am Chem Soc. 2015 Sep 23;137 (37):12100-9. doi: 10.1021/jacs.5b07772. Epub 2015 Sep 15. PMID: 26334259
Frueh DP, Arthanari H, Koglin A, Walsh CT, Wagner G. A double TROSY hNCAnH experiment for efficient assignment of large and challenging proteins. J Am Chem Soc. 2009 Sep 16;131(36):12880-1.
Frueh DP, Arthanari H, Koglin A, Vosburg DA, Bennett AE, Walsh CT, Wagner G. Dynamic thiolation-thioesterase structure of a non-ribosomal peptide synthetase. Nature. 2008 Aug 14;454(7206):903-6.
Selenko, P., Frueh DP, Elsaesser S, Haas W, Gygi SP, Ruderman J, Wagner G, In situ observation of protein phosphorylation by high-resolution NMR spectroscopy. Nat Struct Mol Biol. 2008, 15(3):321-9.
Frueh D, Internal Motions in Proteins and Interference Effects in Nuclear Magnetic Resonance, Prog. NMR spectrosc., 2002, 41, 305-324.