This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The bacterial second messenger bis-(3 '-5 ')-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates complex biological processes such as cell motility, biofilm formation and virulence. Protein domains catalyzing synthesis and degradation of c-di-GMP, GGDEF and EAL domains, respectively, have been identified in large numbers in almost every microbial genome sequenced to date. Our understanding regarding function and regulation of these enzymes is only in its infancy. Using structural biology in combination with enzymatic assays, we hope to decipher the regulatory mechanisms and modes of operation of key enzymes controlling biofilm formation and cytotoxicity, using the opportunistic pathogen Pseudomonas aeruginosa as a model system. Results will have widespread applications such as the development of novel antibiotics for the treatment of infectious diseases, optimization of biotechnological processes, or the development of novel biomedical therapies. In more general terms, we are interested in the intra- and intermolecular regulatory principles in multi-domain proteins, including allosteric and feedback control, fundamental questions in signal transduction and enzyme regulation. We obtained crystals for three proteins containing EAL-, GGDEF- or tandem-domains in various conditions. Preliminary X-ray diffraction experiments performed at CHESS, beamline A1 show promising results. Crystals diffract X-rays to a maximum resolutions of 2.4-10 angstrom but we are confident that improved crystallization and freezing conditions will provide better data quality required for model building and refinement.
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