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

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081324#pone-0081324-t002" target="_blank">Table 2 A)</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>B</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. <b>C</b>) 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). <b>D</b>) Microcalorimetric titrations of 11 μM YfiN_GGDEF with GTP (170 μM in the syringe).