Jimenez Lab

FixL is a heme-based oxygen sensor that regulates nitrogen fixation in rhizobial bacteria. It is a typical histidine kinase containing a PAS motif sensor domain and a histidine kinase domain whose acivity is controlled by oxygen binding in the sensor domain. Crystal structure studies of FixL with and without ligand bound to the heme suggest that conformational changes in the FG loop play an important role in transmitting the signal from the sensor domain to the kinase domain.

Figure 1. Structure of Fe-FixL (1DRM) showing mainly the beta-sheet scaffold that forms a hydrophobic pocket. The FG loop is colored red.

Fluorescence is a useful probe of protein structure and function and has been used in studies of conformational changes, molecular diffusion, protein fluctuation and ligand binding. Unfortunately, the native Fe-proporphyrin IX (PP9) cofactor of FixL is non-fluorescent. However, some metallo-variants of PP9 are fluorescent. Thus, cofactor metal substitution with non-native porphyrins may be used to construct fluorescent analogues of heme-proteins. We recently developed a procedure for reconstituting the heme domain of B. japonicum FixL (BjFixLH) with Mn-, Sn-, Zn- and Co-PP9. Spectra of these samples are shown in Figure 2. Furthermore, we were able to systematically study BjFixLH's stability against denaturation with guanidine hydrochloride (G-HCl) as a function of metal-histidine ligation. The diversity of their stabilities is shown by a comparison of their unfolding titration curves, as plotted in Figure 3.

Figure 2. Absorption spectra of metal substituted BjFixLH (Black: Zn-BjFixLH, Red: Mn-BjFixLH, Blue: Sn-BjFixLH, Green: Co-BjFixLH, Cyan: Fe-BjFixLH).