Rapid Single-Shot Synthesis of the 214 Amino Acid-Long N-Terminal Domain of Pyocin S2
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nihms-1922793.pdf
Description
Published version
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1.08 MB
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Author(s)
Saebi, Azin
Brown, Joseph S
Marando, Victoria M
Hartrampf, Nina
Chumbler, Nicole M
Hanna, Stephanie
Poskus, Mackenzie
Loas, Andrei
Kiessling, Laura L
Hung, Deborah T
Date Issued
February 23, 2023
Journal
ACS Chemical Biology
Publisher
American Chemical Society
Citation
Azin Saebi, Joseph S. Brown, Victoria M. Marando, Nina Hartrampf, Nicole M. Chumbler, Stephanie Hanna, Mackenzie Poskus, Andrei Loas, Laura L. Kiessling, Deborah T. Hung, Bradley L. Pentelute; Rapid Single-Shot Synthesis of the 214 Amino Acid-Long N‑Terminal Domain of Pyocin S2. ACS Chem. Biol. 17 March 2023; 18 (3): 518–527.
Version
Author's final manuscript
Abstract
The impermeable outer membrane of Pseudomonas aeruginosa is bypassed by antibacterial proteins known as S-type pyocins. Because of their properties, pyocins are investigated as a potential new class of antimicrobials against Pseudomonas infections. Their production and modification, however, remain challenging. To address this limitation, we employed automated fast-flow peptide synthesis for the rapid production of a pyocin S2 import domain. The N-terminal domain sequence (PyS2NTD) was synthesized in under 10 h and purified to yield milligram quantities of the desired product. To our knowledge, the 214 amino acid sequence of PyS2NTD is among the longest peptides produced from a “single-shot” synthesis, i.e., made in a single stepwise route without the use of ligation techniques. Biophysical characterization of the PyS2NTD with circular dichroism was consistent with the literature reports. Fluorescently labeled PyS2NTD binds to P. aeruginosa expressing the cognate ferripyoverdine receptor and is taken up into the periplasm. This selective uptake was validated with confocal and super resolution microscopy, flow cytometry, and fluorescence recovery after photobleaching. These modified, synthetic S-type pyocin domains can be used to probe import mechanisms of P. aeruginosa and leveraged to develop selective antimicrobial agents that bypass the outer membrane.
MIT Department
Massachusetts Institute of Technology. Department of Chemistry
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DOI of Published Version
https://doi.org/10.1021/acschembio.2c00862
