Rapid Single-Shot Synthesis of the 214 Amino Acid-Long N-Terminal Domain of Pyocin S2.
Rapid Single-Shot Synthesis of the 214 Amino Acid-Long N-Terminal Domain of Pyocin S2.
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DOI：
10.1021/acschembio.2c00862
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发表时间：
2023-03-17
影响因子：
4
通讯作者：
Pentelute BL
中科院分区：
文献类型：
--
作者：
Saebi A;Brown JS;Marando VM;Hartrampf N;Chumbler NM;Hanna S;Poskus M;Loas A;Kiessling LL;Hung DT;Pentelute BL
被称为S型绿脓菌素的抗菌蛋白绕过铜绿假单胞菌的不可渗透的外膜。由于它们的性质，绿脓菌素被研究作为针对假单胞菌感染的潜在的新类别的抗菌剂。然而，它们的生产和改造仍然具有挑战性。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绿脓菌素S2输入结构域。在10小时内合成N-末端结构域序列（PyS 2NTD），并纯化以产生毫克量的所需产物。据我们所知，PyS 2NTD的214个氨基酸序列是由“单次”合成产生的最长肽之一，即，在不使用连接技术的单一逐步途径中制备。具有圆二色性的PyS 2 NTD的生物物理特征与文献报道一致。荧光标记的PyS 2NTD与表达同源铁绿脓菌荧光素受体的铜绿假单胞菌结合，并被吸收到周质中。这种选择性摄取与共聚焦和超分辨率显微镜，流式细胞术，和光漂白后的荧光恢复进行了验证。这些修饰的合成S型绿脓菌素结构域可用于探测铜绿假单胞菌的输入机制，并用于开发绕过外膜的选择性抗微生物剂。
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.
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影响因子：
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