A simple method to determine changes in the affinity between HisF and HisH in the Imidazole Glycerol Phosphate Synthase heterodimer

by Vitor M. Almeida, J. Patrick Loria, Sandro R. Marana

The bi-enzyme HisF-HisH heterodimer is part of the pathway that produces histidine and purines in bacteria and lower eukaryotes, but it is absent in mammals. This heterodimer has been largely studied probing the basis of the allosteric effects and the structural stability in proteins. It is also a potential target for antibacterial drugs. In this work, we developed a simple method to evaluate changes in the affinity between HisF and HisH in the heterodimer of the bacteria Thermotoga maritima. HisH contains a single tryptophan residue, which is exposed in the free protein, but buried in the heterodimer interface. Hence, the intrinsic fluorescence maximum of this residue changes to shorter wavelengths upon dimerization. Thus, we used the fluorescence intensity at this shorter wavelength to monitor heterodimer accumulation when HisH was combined with sub-stoichiometric HisF. Under conditions where the HisF-HisH heterodimer is in equilibrium with the free states of these enzymes, when [HisH] > [HisF], we deduced a linear function connecting [HisF-HisH] to [HisF], in which the slope depends on the heterodimer dissociation constant (K_d). Based on this equation, taking fluorescence intensities as proxies of the heterodimer and HisF concentrations, we experimentally determined the K_d at four different temperatures. These K_d values were compared to those evaluated using ITC. Both methods revealed an increase in the HisF and HisH binding affinity as the temperature increases. In spite of differences in their absolute values, the K_d determined using these methods presented an evident linear correlation. To demonstrate the effectiveness of the fluorescence method we determined the effect on the K_d caused by 12 single mutations in HisF. Coherently, this test singled out the only mutation in the binding interface. In brief, the method described here effectively probes qualitative effects on the K_d, can be carried out using common laboratory equipment and is scalable.