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Discrimination among protein variants using an unfoldase-coupled nanopore.
- Source :
-
ACS nano [ACS Nano] 2014 Dec 23; Vol. 8 (12), pp. 12365-75. Date of Electronic Publication: 2014 Dec 08. - Publication Year :
- 2014
-
Abstract
- 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.
- Subjects :
- ATPases Associated with Diverse Cellular Activities
Adenosine Triphosphatases metabolism
Endopeptidase Clp metabolism
Escherichia coli Proteins metabolism
Models, Molecular
Molecular Chaperones metabolism
Point Mutation
Protein Stability
Protein Structure, Tertiary
Proteolysis
Nanopores
Nanotechnology instrumentation
Protein Engineering
Protein Unfolding
Proteins chemistry
Proteins genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1936-086X
- Volume :
- 8
- Issue :
- 12
- Database :
- MEDLINE
- Journal :
- ACS nano
- Publication Type :
- Academic Journal
- Accession number :
- 25402970
- Full Text :
- https://doi.org/10.1021/nn5049987