Program in Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto M5G 0A4, Ontario, Canada; Department of Biochemistry, University of Toronto, Toronto M5S 1A8, Ontario, Canada.
Program in Molecular Medicine, Research Institute, Hospital for Sick Children, Toronto M5G 0A4, Ontario, Canada; Department of Biochemistry, University of Toronto, Toronto M5S 1A8, Ontario, Canada.
Biophys J. 2022 Sep 6;121(17):3253-3262. doi: 10.1016/j.bpj.2022.07.026. Epub 2022 Aug 2.
As the bacterial multidrug resistance crisis continues, membrane-active antimicrobial peptides are being explored as an alternate treatment to conventional antibiotics. In contrast to antimicrobial peptides, which function by a nonspecific membrane disruption mechanism, here we describe a series of transmembrane (TM) peptides that are designed to act as drug efflux inhibitors by aligning with and out-competing a conserved TM4-TM4 homodimerization motif within bacterial small multidrug resistance proteins. The peptides contain two terminal tags: a C-terminal lysine tag to direct the peptides toward the negatively charged bacterial membrane, and an uncharged N-terminal sarcosine (N-methyl-glycine) tag to promote membrane insertion. While effective at inhibiting efflux activity, ostensibly through their designed mechanism of action, the impact of the peptides on the bacterial inner membrane remains undetermined. To evaluate the extant peptide-membrane interactions, we performed a series of biophysical measurements. Circular dichroism spectroscopy and Trp fluorescence showed that the peptides insert into the membrane generally in helical form. Interestingly, differential scanning calorimetry of the peptides added to bacterial-like membranes (POPE:POPG 3:1) revealed the peptides' ability to demix the POPE and POPG lipids, creating two pools, one of which is likely a peptide-POPG conglomerate, and the other a POPE-rich component where the native POPG content has been depleted. However, dye leakage assays confirmed that these events occur without causing significant membrane disruption both in vitro and in vivo, indicating that the peptides can target the small multidrug resistance TM4-TM4 motif without nonspecific membrane disruption. In related studies, DiOC(3) fluorescence indicated moderate peptide-mediated reduction of the proton motive force for all peptides, including control peptides that did not display inhibitory activity. The overall findings suggest that peptides designed with suitable tags, sequence hydrophobicity, and charge distribution can be directed more generally to impact proteins whose function involves membrane-embedded protein-protein interactions.
随着细菌的多药耐药性危机的持续，膜活性抗菌肽正被探索作为传统抗生素的替代治疗方法。与通过非特异性膜破坏机制起作用的抗菌肽不同，我们在这里描述了一系列跨膜（TM）肽，这些肽旨在通过与细菌小多重耐药蛋白内的保守 TM4-TM4 同源二聚化基序对齐并与之竞争来作为药物外排抑制剂。这些肽包含两个末端标签：一个 C 末端赖氨酸标签，用于引导肽朝向带负电荷的细菌膜，以及一个不带电荷的 N 末端肌氨酸（N-甲基甘氨酸）标签，以促进膜插入。虽然这些肽通过其设计的作用机制有效地抑制外排活性，但它们对细菌内膜的影响仍未确定。为了评估肽与膜的现存相互作用，我们进行了一系列生物物理测量。圆二色性光谱和色氨酸荧光表明，肽通常以螺旋形式插入膜中。有趣的是，添加到类似细菌的膜（POPE：POPG 3：1）中的肽的差示扫描量热法显示出肽分离 POPE 和 POPG 脂质的能力，形成两个池，其中一个可能是肽-POPG 聚集体，另一个是 POPE 丰富的成分，其中原始的 POPG 含量已耗尽。然而，染料渗漏测定证实，这些事件在体外和体内都不会导致膜的显著破坏，这表明肽可以靶向小多重耐药 TM4-TM4 基序而不会发生非特异性膜破坏。在相关研究中，DiOC（3）荧光表明所有肽都能适度地介导质子动力势的减少，包括没有显示抑制活性的对照肽。总的研究结果表明，设计合适的标签、序列疏水性和电荷分布的肽可以更普遍地靶向其功能涉及膜嵌入式蛋白-蛋白相互作用的蛋白质。
