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, DiOC₂(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.

随着细菌多药耐药性危机的持续，人们正在探索膜活性抗菌肽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发挥作用的抗菌肽相比，我们在此描述了一系列跨膜 (TM) 肽，这些肽旨在通过与内部保守的 TM4-TM4 同二聚化基序对齐并竞争来充当药物外流抑制剂。细菌小多药耐药蛋白。这些肽包含两个末端标签：C 端赖氨酸标签将肽引导至带负电荷的细菌膜，以及不带电荷的 N 端肌氨酸（N-甲基甘氨酸）标签以促进膜插入。虽然表面上通过其设计的作用机制有效抑制外排活性，但肽对细菌内膜的影响仍不确定。为了评估现有的肽-膜相互作用，我们进行了一系列生物物理测量。圆二色光谱和色氨酸荧光显示肽通常以螺旋形式插入膜中。有趣的是，对添加到类细菌膜 (POPE:POPG 3:1) 的肽进行差示扫描量热法揭示了肽能够将 POPE 和 POPG 脂质分层，从而形成两个池，其中之一可能是肽-POPG 复合体，另一个是富含 POPE 的成分，其中天然 POPG 含量已耗尽。然而，染料渗漏测定证实，这些事件的发生在体外和体内均不会引起显着的膜破坏，表明这些肽可以靶向小的多药耐药性 TM4-TM4 基序，而不会造成非特异性膜破坏。在相关研究中，DiOC ₂ (3) 荧光表明所有肽（包括不表现出抑制活性的对照肽）的质子动力都有适度的肽介导的降低。总体研究结果表明，设计有合适标签、序列疏水性和电荷分布的肽可以更广泛地针对其功能涉及膜嵌入蛋白质-蛋白质相互作用的影响蛋白质。