Computational nanopore redesign for the sensing of chiral peptide isomers

在纳米孔感测应用的广泛组合中，手性肽同种型的鉴定是重要的，例如与疾病诊断和肽治疗剂的对映体纯度的阐明相关。使用标准生物分析和色谱技术区分肽的对映异构体异构体是复杂、昂贵和耗时的。纳米孔传感提供了一种便携、廉价和快速检测手性肽的可能性。手性分子的纳米孔传感的早期研究依赖于引入手性部分以使一种手性形式的检测相对于另一种手性形式偏置。最近对肽的研究表明，肽-纳米孔相互作用的差异，以及因此肽同种型的亲和力和动力学的差异，可以产生特征电流信号。基于这些发现，我们假设对于给定的纳米孔，可以设计一组突变，其可以以不同的构象和亲和力结合肽的D和L同种型-这样的突变纳米孔原则上应该为两种同种型产生不同的电流。为了达到这样的纳米孔设计，我们建议开发一种计算纳米孔重新设计流水线，该流水线将Rosetta蛋白质重新设计算法与用于估计开孔和阻断电流的快速分析方法相结合，以生成计算预测为稳定的纳米孔设计，并为肽同种型产生不同的信号。在该方案中，计算设计算法将用于同时优化两种肽同种型的纳米孔-肽相互作用，以使具有不同亲和力和构象的肽能够结合。对于开孔和堵塞电流的估计，我们计划采用最近开发的空间排阻模型，使有前途的设计筛选后，连续几轮的计算设计优化。在我们之前的工作中，我们研究了OmpF纳米孔用于检测五肽的对映体形式，证明了孔用于手性传感的潜力。在拟议的项目中，我们计划研究相同的孔（及其同系物）以及类似的肽，目的是通过应用拟议的计算管道来改善肽的离子电流识别，从而能够与现有的实验数据进行直接比较。拟议工作的一个主要部分将集中在开发和测试的设计管道和目前的估计程序与比较的数据从全原子MD模拟在适当的情况下。总之，我们期望得到一组潜在的纳米孔突变体，其允许明确区分不同的同种型，并且可以用作这种纳米孔的实验测试的基础。

n the broad portfolio of nanopore sensing applications, the identification of chiral peptide isoforms is an important one, relevant for example to disease diagnostics and the elucidation of the enantiomeric purity of peptide therapeutics. Differentiating enantiomeric isoforms of peptides using standard bioanalytical and chromatographic techniques is both complicated, expensive, and time-consuming. Nanopore sensing offers the possibility of a portable, inexpensive, and rapid detection of chiral peptides. Early studies of nanopore sensing of chiral molecules relied on the introduction of chiral moieties to bias the detection of one chiral form over the other. More recent work on peptides has shown that differences in the peptide-nanopore interactions, and consequently in the affinities and kinetics of peptide isoforms, can give rise to characteristic current signals. Based on these findings, we hypothesize that for a given nanopore one could design a set of mutations that can bind the D and L isoforms of a peptide with distinct conformations and affinities – such a mutant nanopore should in principle generate different currents for the two isoforms. To arrive at such a nanopore design, we propose to develop a computational nanopore redesign pipeline that combines the Rosetta protein redesign algorithms with a fast analytical method for estimating the open pore and blockade currents to generate nanopore designs that are computationally predicted to be stable and produce a distinct signal for the peptide isoforms. Within this protocol, the computational design algorithm would serve to simultaneously optimize the nanopore-peptide interactions of both peptide isoforms to enable the binding of the peptides with different affinities and conformations. For the estimation of the open pore and blockage currents, we plan to employ the recently developed steric exclusion model enabling the screening for promising designs after successive rounds of computational design optimization. In our previous work, we had investigated the OmpF nanopore for the detection of the enantiomeric forms of a pentapeptide, demonstrating the potential of the pore for chirality sensing. In the proposed project, we plan to investigate the same pore (and its homologues) together with similar peptides, with the goal of improving the ionic current discrimination of the peptides through the application of the proposed computational pipeline, enabling a straightforward comparison with the available experimental data. A major part of the proposed work would focus on the development and testing of the design pipeline and the current estimation procedure with comparisons to data from all-atom MD simulations where appropriate. In conclusion, we expect to arrive at a set of potential nanopore mutants that allow a clear discrimination of different isoforms and that can be used as base for the experimental testing of such nanopores.