Figure 4. Binding affinity for nucleotides and enzymatic activity of YfiN_HAMP-GGDEF and YfiN_GGDEF.

For all ITC experiments upper panels show the Raw ITC data, while lower panels show the integrated peak areas (black square) fitted with the one-binding-site model of ORIGIN provided by MicroCal (continuous lines). Derived thermodynamic parameters are listed in Table 2 A) Microcalorimetric titration of 3 μM YfiN_HAMP-GGDEF with c-di-GMP (90 μM in the syringe). No binding was observed either in the presence of CaCl₂ or in the presence of MgCl₂/MnCl₂ (data not shown). No thermodynamic parameters were derived. B) Microcalorimetric titrations of 14 μM enzyme solution with GTP (170 μM in the syringe). The thermodynamic profile indicates that the interaction of YfiN_HAMP-GGDEF with GTP presents favorable binding enthalpy and entropy, which suggests that hydrogen bonding and hydrophobic interactions are mainly involved in the binding event, rather than conformational changes. C) Cyclase activity of 10 µM YfiN_HAMP-GGDEF or YfiN_GGDEF assayed in real time by circular dichroism spectroscopy after addition of 100 µM GTP. For YfiN_HAMP-GGDEF (Black) The final c-di-GMP concentration corresponds to complete conversion of 100 µM GTP, whilst for YfiN_GGDEF (grey) no product is detected even if the sample is allowed to react for 24 h (not shown). D) Microcalorimetric titrations of 11 μM YfiN_GGDEF with GTP (170 μM in the syringe).