Rapid Single-Shot Synthesis of the 214 Amino Acid-Long N-Terminal Domain of Pyocin S2,ACS Chemical Biology

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Rapid Single-Shot Synthesis of the 214 Amino Acid-Long N-Terminal Domain of Pyocin S2
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2023-02-23 , DOI: 10.1021/acschembio.2c00862
Azin Saebi ₁ , Joseph S Brown ₁ , Victoria M Marando ₁ , Nina Hartrampf ₁ , Nicole M Chumbler _{2,

3,

4} , Stephanie Hanna ₁ , Mackenzie Poskus ₁ , Andrei Loas ₁ , Laura L Kiessling ₁ , Deborah T Hung _{2,

3,

4} , Bradley L Pentelute _{1,

2,

5,

6}

Affiliation

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 (PyS2^NTD) 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 PyS2^NTD 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 PyS2^NTD with circular dichroism was consistent with the literature reports. Fluorescently labeled PyS2^NTD 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.

中文翻译：

快速单次合成 Pyocin S2 的 214 个氨基酸长 N 端结构域

铜绿假单胞菌不可渗透的外膜被称为 S 型脓毒素的抗菌蛋白绕过。由于其特性，脓毒素被研究为一种潜在的新型抗假单胞菌感染抗菌剂。然而，它们的生产和修改仍然具有挑战性。为了解决这一限制，我们采用自动化快速流程肽合成来快速生产化脓菌素 S2 导入结构域。N 端结构域序列 (PyS2 ^NTD ) 在 10 小时内合成并纯化，产生毫克量的所需产物。^{据我们所知，PyS2 NTD}的 214 个氨基酸序列是通过“单次”合成产生的最长的肽之一，即在不使用连接技术的情况下以单一逐步途径制备的肽。具有圆二色性的 PyS2 ^NTD的生物物理表征与文献报道一致。荧光标记的 PyS2 ^{NTD与表达同源}铁吡维丁受体的铜绿假单胞菌结合，并被吸收到周质中。这种选择性摄取通过共聚焦和超分辨率显微镜、流式细胞术以及光漂白后的荧光恢复进行了验证。这些修饰的合成 S 型脓毒素结构域可用于探测铜绿假单胞菌的输入机制，并可用于开发绕过外膜的选择性抗菌剂。

更新日期：2023-02-23

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