This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Environmental signals that trigger bacterial pathogenesis and biofilm formation are mediated by changes in the level of cyclic dimeric guanosine monophosphate (c-di-GMP), a unique eubacterial second messenger. Tight regulation of cellular c-di-GMP domain concentration is governed by diguanylate cyclases and phosphadiesterases, which are responsible for its production and degradation, respectively. Diguanylate cyclase WspR is a conserved GGDEF domain-containing response regulator in Gram-negative bacteria and it is bound to c-di-GMP at an inhibitory site. Overexpression of WspR causes hyperbiofilm formation, whereas loss-of-function mutants show reduced biofilm formation and cytotoxicity. The crystal structure of full length Diguanylate cyclase WspR from P. aeruginosa in its inhibitory state was recently resolved in the group of Professor Holger Sondermann, Cornell University. However, X-ray crystallography experiences difficulties to study the oligomeric states of WspR that appear in solution since at high concentration, used in such an experiment, the protein spontaneously forms tetramer. We used pulsed dipolar ESR spectroscopy (PDS) to elucidate the structure of differenr oligomeric (functional) states of Diguanylate Cyclate WspR. Paramagnetic spin-labels (MTSSL) were attached to naturally accruing cysteine residues in different domains of a single protein molecule. A single WspR molecule contains two cysteine residues at positions 49 in CheY domain and 240 in GGDEF domain. In order to reduce the number of spin-labels in the dimeric and tetrameric state of this protein and consequently to avoid a complication in the interpretation of results, two alternative point mutations were done: C49S or C240S, and distances between spin-labels attached to the remaining cysteines were measured.
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