Dataset for: Dynamic Gating by the Phenylalanine Clamp Loop Controls Peptide Translocation Through the Anthrax Toxin Nanopore

The ϕ-clamp loop, which contains the key F427 residue, is a critical active site in the anthrax toxin protective antigen (PA) nanopore, yet its precise role in governing the complex, multi-state dynamics of peptide translocation remains debated. Here, we dissect the peptide-clamp interaction mechanism using single-channel electrophysiology and a series of guest-host peptides, which are translocated via either wild-type PA, an ablated F427A mutant, or a polar aromatic F427Y mutant. Mutations to the ϕ clamp dramatically reduce peptide residence times but, critically, preserve the intermediate, partially blocked conductance states observed in the wild-type pore. Thermodynamic analysis reveals that the F427 residue is essential for creating a deep, energetically stable, fully blocked ‘hydrophobic trap’ (State 0), as its mutation leads to a significant destabilization of this state and a corresponding population shift to shallower intermediates. Kinetic analysis of the state-to-state transitions demonstrates that while the F427A mutation lowers the energetic barrier for escape from this trap, it disrupts the efficient, hydrophobically driven entry. Furthermore, the strong correlations between kinetic parameters and peptide molecular properties (hydrophobicity, aromaticity) that are a hallmark of the wild-type pore are completely abolished in the F427A mutant. These results support a refined model where F427 acts as a specific chemical ‘reader,’ and the intermediate states arise from larger-scale, dynamic dilation of the entire clamp-containing loop. This detailed mechanistic insight provides a framework for the rational engineering of next-generation nanopore biosensors.Φ夹环（phi-clamp loop）包含关键残基F427，是炭疽毒素保护性抗原（protective antigen, PA）纳米孔的关键活性位点，但其在调控肽易位的复杂多态动力学过程中的精确作用仍存在争议。本研究采用单通道电生理学与一系列客-宿主肽（guest-host peptides），分别通过野生型PA、F427A突变体以及极性芳香族F427Y突变体介导肽易位，剖析肽-夹环相互作用机制。对Φ夹环的突变可显著缩短肽的驻留时间，但关键在于，其保留了野生型孔道中观测到的部分阻断型中间电导状态。热力学分析表明，F427残基对于形成深层、能量稳定的完全阻断型“疏水陷阱（hydrophobic trap）”（状态0）至关重要，因为该残基的突变会显著削弱该状态的稳定性，并使态占据分布相应向更浅的中间态偏移。对态间跃迁的动力学分析表明，尽管F427A突变降低了肽从该疏水陷阱逃逸的能垒，但却破坏了由疏水作用驱动的高效进入过程。此外，野生型孔道标志性的动力学参数与肽分子特性（疏水性、芳香性）之间的强相关性，在F427A突变体中完全消失。上述结果支持一个改良模型，其中F427充当特异性化学“阅读器”，而中间态则源自整个含夹环区域的大规模动态扩张。这一详尽的机制性见解为下一代纳米孔生物传感器的理性设计提供了理论框架。