Asymmetric Single-Chain MspA nanopores for electroosmotic stretching and sequencing proteins

项目总结/摘要 从微量中进行蛋白质鉴定和/或单分子蛋白质测序可以彻底改变我们的 通过提供细胞在其最高水平上的分子状态的图片来了解健康 功能分子通过这个多PI提案，我们将开发原型工具， 基于蛋白质的纳米孔，用于探测单个蛋白质分子的序列。蛋白质测序工具 是基于工程MspA，一种来自耻垢分枝杆菌的孔蛋白，已用于纳米孔， 基于DNA测序。然而，我们的蛋白质检测/测序工具将在以下方面进行工程设计 方式：1）构成八聚体组装体的每个蛋白质单体共价连接， 能够沿着这种单链MspA（scMspA）蛋白进行诱变，以产生一系列突变体， 作为用于阅读氨基酸序列的高分辨率纳米孔的不对称收缩（最近 Niederweis和Wanunu小组证明），2）DNA加工酶将用于移动短距离 肽和全长蛋白质通过这些肽与DNA的缀合和ATP介导的 DNA易位，3）我们的系统与孔隙期间变性电解质条件的完全兼容性 实验将促进序列中高精度肽/蛋白质读出的三个关键要求- 独立的方式-蛋白质解折叠，蛋白质穿线，以及拉伸蛋白质的驱动力， 在毛孔处被拉紧（所有这些都是最近由Wanunu，Aksimentiev和Chen小组证明的）。这些 结合创新，结合团队的关键技术能力，将使我们能够开发出一种 蛋白质测序原型。我们建议通过三个主要目标的研究来实现我们的目标：1）我们 将设计和测试各种不对称scMspA突变体，以优化类似蛋白的信号对比度 序列与单个氨基酸取代，2）我们将证明解旋酶介导的运动肽 通过scMspA突变体和信号解码构建文库，以及3）我们将读取全长未折叠的 蛋白质，并训练模型基于以下内容识别这些集合： 纯蛋白质样品。对于最有希望的突变体scMspA，我们将目标定为>90%的区分准确率。 在样品组中的所有肽/蛋白质中。我们开发的平台的成功将导致采用和 产品开发，以彻底改变单分子和单细胞蛋白质组学。

Project Summary / Abstract Protein identification and/or single-molecule protein sequencing from minute amounts could revolutionize our understanding of health by providing a picture of the molecular state of the cell at the level of its most functional molecules. Through this multi-PI proposal, we will develop prototype tools that employ innovative protein-based nanopores for probing the sequence of individual protein molecules. The protein sequencing tool is based on engineered MspA, a porin from Mycobacterium smegmatis that has been utilized in nanopore- based DNA sequencing. However, our protein detection/sequencing tool will be engineered in the following ways: 1) Each of the protein monomers that comprise the octameric assembly are covalently connected, enabling mutagenesis along this single-chain MspA (scMspA) protein to create a series of mutants with asymmetric constrictions as high-resolution nanopores for reading amino acid sequences (recently demonstrated by Niederweis and Wanunu groups), 2) A DNA-processing enzyme will be used to move short peptides and full-length proteins through the pore by conjugation of these peptides to DNA and ATP-mediated DNA translocation, 3) the full compatibility of our system with denaturing electrolyte conditions during pore experiments will facilitate three critical requirements for high accuracy peptide/protein readout in a sequence- independent manner - protein unfolding, protein threading, and a driving force to stretch the protein so it is pulled taut at the pore (all of these recently demonstrated by Wanunu, Aksimentiev, and Chen groups). These combined innovations, combined with key technological capabilities of the team, will allow us to develop a protein sequencing prototype. We propose to achieve our goals through research in three main aims: 1) We will engineer and test various asymmetric scMspA mutants to optimize signal contrast from similar protein sequences with single amino acid substitutions, 2) we will demonstrate helicase-mediated motion of peptide libraries through scMspA mutants and signal decoding, and 3) we will read subsets of full-length unfolded proteins in a complex sample that contains many proteins, and train a model to recognize this sets based on pure protein samples. For the most promising mutant scMspA we will target >90% accuracy in distinguishing among all peptides/proteins in the sample set. Success in our developed platform will result in adoption and product development in order to revolutionize single-molecule and single-cell proteomics.