Dataset for: Comparative study of nanopore phenylalanine clamp variants reveals unique peptide biosensing and classification properties

Abstract Rationally engineering biological nanopores is critical for advancing single-molecule biosensing. Here, we investigate the phenylalanine clamp active site (ϕ clamp) of the anthrax toxin protective antigen (PA) nanopore, a key site for molecular interaction, to test if engineering this site can improve peptide classification. We performed a comparative analysis of wild-type PA and two ϕ-clamp mutants (F427A, F427Y). We report the paradoxical finding that the F427A mutant—known to be a defective large protein translocase—is a superior peptide biosensor. Using a machine learning framework with engineered biophysical features, the F427A pore classifies a diverse peptide set with 93% accuracy. Our analysis suggests this enhanced performance arises because the F427A mutation, while weakening specific interactions, produces more consistent, lower-variance kinetic ‘fingerprints’ that are more easily distinguished by computational models. These findings establish a principle for biosensor design and enable a strategy where engineered pores with complementary specificities are deployed in multiplexed arrays for robust diagnostics.摘要 理性工程化改造生物纳米孔(biological nanopores)，是推动单分子生物传感技术发展的关键所在。本研究聚焦炭疽毒素保护性抗原(anthrax toxin protective antigen, PA)纳米孔的苯丙氨酸钳活性位点（ϕ钳）——这一核心分子相互作用位点——开展探究，以验证对该位点进行工程化改造能否提升肽段分类性能。 我们针对野生型PA与两种ϕ钳突变体（F427A、F427Y）完成了对比分析。研究得到一项悖谬性发现：此前被认定存在功能缺陷的大型蛋白质转位酶F427A突变体，实际却是性能更优异的肽段生物传感器。借助集成工程化生物物理特征的机器学习框架，F427A纳米孔可对多样化肽段数据集实现93%的分类准确率。 分析表明，该性能提升源于F427A突变的独特效应：尽管该突变削弱了部分特异性相互作用，却产生了一致性更强、方差更低的动力学“指纹”特征，使得计算模型更易实现区分。本研究成果确立了生物传感器设计的一项通用原则，并提出了一种可行策略：将具备互补特异性的工程化纳米孔部署于多重阵列中，以实现稳定可靠的诊断检测。