<br>用于下一代抗生素研究的基本 ABC 转运蛋白多模式表征的生物传感器,ACS Applied Materials & Interfaces

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用于下一代抗生素研究的基本 ABC 转运蛋白多模式表征的生物传感器
ACS Applied Materials & Interfaces ( IF 8.3 ) Pub Date : 2023-03-03 , DOI: 10.1021/acsami.2c21556
Karan Bali ₁ , Charlotte Guffick ₂ , Reece McCoy ₁ , Zixuan Lu ₁ , Clemens F Kaminski ₁ , Ioanna Mela ₁ , Róisín M Owens ₁ , Hendrik W van Veen ₂

Affiliation

随着抗生素耐药性威胁的增加，人们特别关注开发针对病原菌的抗菌药物，这些病原菌的多重耐药性尤其根深蒂固且令人担忧。新型抗菌药物的此类靶标之一是 ATP 结合盒 (ABC) 转运蛋白 MsbA，它存在于革兰氏阴性病原菌的质膜中，对于这些细菌的生存至关重要。支持的脂质双层 (SLB) 可用于监测膜蛋白结构和功能，因为它们可以与各种光学、生化和电化学技术集成。在这里，我们形成了含有大肠杆菌MsbA 的 SLB，并使用原子力显微镜 (AFM) 和结构照明显微镜 (SIM) 作为高分辨率显微镜技术来研究 SLB 和掺入的 MsbA 蛋白的完整性。然后，我们将这些 SLB 集成到基于导电聚合物聚（3,4-乙烯二氧基噻吩）聚（苯乙烯磺酸）（PEDOT:PSS）的微电极阵列（MEA）上，使用电化学阻抗谱（EIS）监测流经 MsbA 蛋白的离子流响应 ATP 水解。这些 EIS 测量可与 MsbA-ATPase 活性的生化检测相关。为了展示这种 SLB 方法的潜力，我们不仅观察了野生型 MsbA 的活性，还观察了两个先前表征的突变体以及基于喹啉的 MsbA 抑制剂 G907 的活性，以表明 EIS 系统可以检测 ABC 转运蛋白活性的变化。我们的工作结合了多种技术来彻底研究脂质双层中的 MsbA 以及该蛋白质的潜在抑制剂的作用。我们预计该平台将促进抑制 MsbA 或微生物中其他重要膜转运蛋白的下一代抗菌药物的开发。

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Biosensor for Multimodal Characterization of an Essential ABC Transporter for Next-Generation Antibiotic Research

As the threat of antibiotic resistance increases, there is a particular focus on developing antimicrobials against pathogenic bacteria whose multidrug resistance is especially entrenched and concerning. One such target for novel antimicrobials is the ATP-binding cassette (ABC) transporter MsbA that is present in the plasma membrane of Gram-negative pathogenic bacteria where it is fundamental to the survival of these bacteria. Supported lipid bilayers (SLBs) are useful in monitoring membrane protein structure and function since they can be integrated with a variety of optical, biochemical, and electrochemical techniques. Here, we form SLBs containing Escherichia coli MsbA and use atomic force microscopy (AFM) and structured illumination microscopy (SIM) as high-resolution microscopy techniques to study the integrity of the SLBs and incorporated MsbA proteins. We then integrate these SLBs on microelectrode arrays (MEA) based on the conducting polymer poly(3,4-ethylenedioxy-thiophene) poly(styrene sulfonate) (PEDOT:PSS) using electrochemical impedance spectroscopy (EIS) to monitor ion flow through MsbA proteins in response to ATP hydrolysis. These EIS measurements can be correlated with the biochemical detection of MsbA-ATPase activity. To show the potential of this SLB approach, we observe not only the activity of wild-type MsbA but also the activity of two previously characterized mutants along with quinoline-based MsbA inhibitor G907 to show that EIS systems can detect changes in ABC transporter activity. Our work combines a multitude of techniques to thoroughly investigate MsbA in lipid bilayers as well as the effects of potential inhibitors of this protein. We envisage that this platform will facilitate the development of next-generation antimicrobials that inhibit MsbA or other essential membrane transporters in microorganisms.

更新日期：2023-03-03

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