Interaction study of the Pb27 protein from Paracoccidioides brasiliensis with a nucleotide ligand and its application in a biosensor

The protein Pb27 from Paracoccidioides brasiliensis is an antigen that has been studied for the production of vaccines and in the diagnosis of Paracoccidioidomycosis (PCM), the main systemic mycosis in Latin America, which epidemiological importance is due to its extensive area of endemicity in Brazil, and to the involvement of active age rural workers. Recently, the fist structure model of Pb27 was determined by X-ray diffraction crystallography, and by structural homology studies the protein was classified as a nucleotidyltransferase of unknown function. In addition, virtual ligand screening and nuclear magnetic resonance studies have identified cytidine triphosphate (CTP) nucleotide as a likely protein ligand. In order to characterize quantitatively Pb27's interaction with this nucleotide, interaction assays were performed by quenching of tryptophan fluorescence, differential scanning fluorimetry and isothermal titration calorimetry. The results indicated a weak interaction, with a dissociation constant of 10-3 M order, exothermic (ΔH = -34,3 KJ/mol) and exergonic (-15,9 KJ/mol). Moreover, we carried out preliminary studies on the protein's use in detection of anti-Pb27 antibodies in an impedimetric biosensor, for future use in PCM diagnosis. The results indicated the recognition of antibodies by the protein, by electrochemical impedance spectroscopy analysis. However, more studies are still needed for the standardization of biosensors, and future tests with patient sera. (author)