ZorO is a ribosomally encoded small protein that serves as the toxin of a toxin-antitoxin locus found in pathogenic strains of <i>Escherichia coli</i>. While toxicity is observed when ZorO is highly expressed, the biological function of this molecule is unknown. Preliminary studies indicate that the function likely depends on its membrane localization and/or oligomerization. The peptide “toxin” may be creating membrane-spanning ion channels that disrupt the proton gradient or embedding in membranes and acting as a protein recruiter. To probe the function and localization of ZorO, native and modified peptides were synthesized via solid phase peptide synthesis using the fluorenylmethyloxycarbonyl chloride/tert-butyl approach. Initially, native ZorO was successfully synthesized. Photo-methionine, a derivative of methionine which permits photo-crosslinking, was then synthesized and incorporated into a ZorO analogue intended for capturing transient protein interactions. A ZorO mutant containing a 1X-FLAG tag was also synthesized, allowing for affinity purification and detection by western blotting. Full scan ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) were used to characterize the synthetic peptides and provide partial sequencing information. The synthetic peptides will be used for liposome and <i>in vitro</i> studies aimed at elucidating the function of ZorO.

Synthesis of modified ZorO analogues for probing biological function

255th National Meeting (2018)

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