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

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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.