Discrimination among protein variants using an unfoldase-coupled nanopore.
Previously we showed that the protein unfoldase ClpX could facilitate translocation of individual proteins through the α-hemolysin nanopore. This results in ionic current fluctuations that correlate with unfolding and passage of intact protein strands through the pore lumen. It is plausible that this technology could be used to identify protein domains and structural modifications at the single-molecule level that arise from subtle changes in primary amino acid sequence (e.g., point mutations). As a test, we engineered proteins bearing well-characterized domains connected in series along an ∼700 amino acid strand. Point mutations in a titin immunoglobulin domain (titin I27) and point mutations, proteolytic cleavage, and rearrangement of beta-strands in green fluorescent protein (GFP), caused ionic current pattern changes for single strands predicted by bulk phase and force spectroscopy experiments. Among these variants, individual proteins could be classified at 86-99% accuracy using standard machine learning tools. We conclude that a ClpXP-nanopore device can discriminate among distinct protein domains, and that sequence-dependent variations within those domains are detectable.
Nanopore Group, Department of Biomolecular Engineering, University of California , Santa Cruz, California 95064, United States.
Jeff
J
Nivala
Logan
L
Mulroney
Gabriel
G
Li
Jacob
J
Schreiber
Mark
M
Akeson
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000378:metabolism; D049071:Endopeptidase Clp / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism; D058608:Nanopores; D036103:Nanotechnology / Q000295:instrumentation*; D017354:Point Mutation; D015202:Protein Engineering; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics*; D059748:Proteolysis
D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D011506:Proteins; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities
nanopore; protein sequencing; protein translocation; single-molecule; unfoldase; α-hemolysin