The Gram negative flagellate Vibrio cholerae survives between cholera epidemics as surface biofilms in aquatic bodies. These surface biofilms are resistant to external stress like antibiotics, chlorine, predators and other factors [1, 2]. Proteins with GGEEF domain have diguanylate cyclase activity which modulates the intracellular level of bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) which, in turn, regulates the switch between the motile and planktonic form of the pathogen. VC0395_0300 is a putative protein from Vibrio cholerae that has the GGEEF domain which shows promising biofilm forming capability. Constructs of the gene-of-interest were created to provide structural and functional insights into the role of the GGEEF domain in biofilm formation. The structure was modeled by using closest structural homolog template and maximum sequence alignment. Comparison with available crystal structures of the GGEEF domain depicted the presence of a central β sheet and five α-helices. This is supported by secondary structure prediction which shows 41.43% helix, 23.36% strand and 35.20% loops in the protein structure.
The presence of tryptophan residues (W4 and W172) in the proteins has enabled us to monitor tryptophan fluorescence by thermal and chemically induced unfolding. From the fluorescence quenching studies with acrylamide, KI and CsCl on native and denatured protein, we could determine that one of the tryptophans resides in a positively charged pocket. Furthermore, transverse urea gradient gel (TUGE) reinforces the fact that the protein shows two-state (native and unfolded) unfolding transition. Likewise, size exclusion chromatography and glutaraldehyde cross-linking have highlighted the oligomerization tendency of the protein. To gather more information on the crystal structure, crystallization trials were attempted in various screens and the work is in progress.
The authors would like to acknowledge BRNS, DAE for funding the research project and for providing fellowship to DB.