Protocol for design, construction, and selection of genome phage (gPhage) display libraries.

This protocol describes the genomic phage (gPhage) display platform, a large-scale antigen and epitope mapping technique. We constructed a gPhage display peptide library of a eukaryotic organism, Trypanosoma cruzi (causative agent of Chagas disease), to map the antibody response landscape against the parasite. Here, we used an organism with a relatively large but intronless genome, although future applications could include other prevalent or (re)emerging infectious organisms carrying genomes with a limited number of introns. For complete details on the use and execution of this protocol, please refer to Teixeira et al. (2021).

Synthesis and interaction of terminal unsaturated chemical probes with Mycobacterium tuberculosis CYP124A1.

A series of C15-C20 isoprenyl derivatives bearing terminal alkenyl and alkynyl groups were synthesized as possible substrates of the methyl-branched lipid ω-hydroxylase CYP124A1 from Mycobacterium tuberculosis. The interactions of each compound with the enzyme active site were characterized using UV-vis spectroscopy. We found that C10 and C15 analogs bind with similar affinity to the corresponding parent C10 and C15 substrates geraniol and farnesol, respectively. Three analogs (C10-ω-ene, C10-ω-yne, C15-ω-yne) interact with the proximal side of the heme iron by coordinating to the oxygen atom of the ferric heme, as judged by the appearance of typical Type-IA binding spectra. On the other hand, the C15-ω-ene analog interacts with the ferric heme by displacing the bound water that generates a typical Type I binding spectrum. We were unable to detect P450-mediated oxidation of these probes following extended incubations with CYP124A1 in our reconstituted assay system, whereas a control reaction containing farnesol was converted to ω-hydroxy farnesol under the same conditions. To understand the lack of detectable oxidation, we explored the possibility that the analogs were acting as mechanism-based inhibitors, but we were unable to detect time-dependent loss of enzymatic activity. In order to gain insight into the lack of detectable turnover or time-dependent inhibition, we examined the interaction of each compound with the CYP124A1 active site using molecular docking simulations. The docking studies revealed a binding mode where the terminal unsaturated functional groups were sequestered within the methyl-binding pocket, rather than positioned close to the heme iron for oxidation. These results aid in the design of specific inhibitors of Mtb-CYP124A1, an interesting enzyme that is implicated in the oxidation of methyl-branched lipids, including cholesterol, within a deadly human pathogen.