Enhancing the Zn-Sequestering Ability of Calprotectin by Engineered Mutation
Calprotectin (CP) is a Ca2 binding protein involved in metal sequestration from microbial pathogens in a host-defense mechanism called nutritional immunity. There are two transition-metal-binding sites within the heterodimer arrangement of S100A8 and S100A9 proteins that make up calprotectin which are pivotal in this antimicrobial response. Due to the existence of pathogens that can pirate the metal chelated by calprotectin resulting in exacerbated disease, a way to increase the affinity of calprotectin to transition metals was researched. This study designed a mutation in the S100A9 protein that changed the aspartic acid on the 30th amino acid to a cysteine (D30C) in an effort to strengthen the ability of CP to chelate metals from microbes. Engineering of the D30C mutation was executed by primer design and mutagenesis. Miniprep and sequencing were vital in verifying the presence of the mutation. CP was then purified by anion exchange chromatography and size exclusion chromatography and quantified by UV-Vis spectroscopy. Upon characterization of CP using isothermal titration calorimetry to analyze whether CP bound zinc at a higher affinity, the intensity of the binding bordered the limits of the capabilities of the instrument and there were two inflection points, posited to be caused by separate binding events from the two binding sites. Thus, the first transition-metal-binding site of CP was knocked out (Δ1) and a series of fluorescence chelator competition assays were performed to characterize the equilibrium inhibition coefficient Ki in order to determine if the mutant did increase metal affinity.