Our studies are intended to improve molecular understanding of NRPS enzymology with the ultimate goal of NRPS reengineering to produce novel complex molecules as potential therapeutics to address difficult medical problems. Nonribosomal peptide synthetases (NRPSs) are large multimodular enzymes in bacteria and fungi that catalyze complex reactions to assemble structurally and functionally diverse bioactive natural products. NRPSs are comprised of several distinct modular units, which can be further divided into catalytic domains involved in substrate activation, peptide bond formation, and product release.The large number of conformational states available to NRPS associated proteins make them demanding targets for structure determination to achieve more complete view of the mechanisms underlying NRP synthesis. However, the sheer size as well as enormous flexibility of NRPS domains poses significant experimental challenges. Specifically designed and synthesized chemical probes aim to freeze highly transient multidomain complexes in biologically relevant conformations, thus yielding X-ray quality protein crystals.Crystallographic data analysis, performed in a computer lab, in turn provides in depth understanding of protein dynamics, determinants of specificity (DoS), mechanistic aspects of transformations performed by studied domains, and more.In addition to the core catalytic domains, many auxiliary proteins with yet undefined purposiveness or mechanism exist in NRPS gene clusters. Identification and characterization of such proteins are of high importance since many of them form functionally relevant interactions with the core NRPS domains. These protein-protein interactions are the object of interest.Using modern analytical tools such as fast protein liquid chromatography (FPLC), isothermal titration calorimetry (ITC), circular dichroism spectroscopy (CD) allows monitoring and evaluation of processes associated with formation of protein complexes (i.e. protein conformations, structural changes, thermodynamics of binding). Attempts to crystallize these complexes should at the end provide structural rationale for observed binding modes.
The large number of conformational states available to NRPS associated proteins make them demanding targets for structure determination to achieve more complete view of the mechanisms underlying NRP synthesis. However, the sheer size as well as enormous flexibility of NRPS domains poses significant experimental challenges. Specifically designed and synthesized chemical probes aim to freeze highly transient multidomain complexes in biologically relevant conformations, thus yielding X-ray quality protein crystals.
Crystallographic data analysis, performed in a computer lab, in turn provides in depth understanding of protein dynamics, determinants of specificity (DoS), mechanistic aspects of transformations performed by studied domains, and more.
In addition to the core catalytic domains, many auxiliary proteins with yet undefined purposiveness or mechanism exist in NRPS gene clusters. Identification and characterization of such proteins are of high importance since many of them form functionally relevant interactions with the core NRPS domains. These protein-protein interactions are the object of interest.
Using modern analytical tools such as fast protein liquid chromatography (FPLC), isothermal titration calorimetry (ITC), circular dichroism spectroscopy (CD) allows monitoring and evaluation of processes associated with formation of protein complexes (i.e. protein conformations, structural changes, thermodynamics of binding). Attempts to crystallize these complexes should at the end provide structural rationale for observed binding modes.