Abstract
Manipulating single molecules and systems of molecules with mechanical force is a powerful technique to examine their physical properties. Applying force requires attachment of the target molecule to larger objects using some sort of molecular tether, such as a strand of DNA. DNA handle attachment often requires difficult manipulations of the target molecule, which can preclude attachment to unstable, hard to obtain, and/or large, complex targets. Here we describe a method for covalent DNA handle attachment to proteins that simply requires the addition of a preprepared reagent to the protein and a short incubation. The handle attachment method developed here provides a facile approach for studying the biomechanics of biological systems.
Keywords:
SpyCatcher; SpyTag; forced unfolding; magnetic tweezers; membrane protein; optical tweezers; protein folding.
© 2016 The Protein Society.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Amino Acid Sequence
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Biomechanical Phenomena
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Cloning, Molecular
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DNA / chemistry*
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DNA-Binding Proteins / chemistry*
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism
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Dimyristoylphosphatidylcholine / chemistry
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Endopeptidases / chemistry*
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Endopeptidases / genetics
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Endopeptidases / metabolism
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Escherichia coli Proteins / chemistry*
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / metabolism
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Gene Expression
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Lipid Bilayers / chemistry
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Magnets
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Maltose-Binding Proteins / chemistry*
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Maltose-Binding Proteins / genetics
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Maltose-Binding Proteins / metabolism
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Membrane Proteins / chemistry*
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Optical Tweezers
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Peptides / chemical synthesis
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Peptides / chemistry
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Peptides / metabolism
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Protein Engineering / methods*
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Protein Folding
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Recombinant Fusion Proteins / chemistry*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Staining and Labeling / methods*
Substances
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DNA-Binding Proteins
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Escherichia coli Proteins
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GlpG protein, E coli
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Lipid Bilayers
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Maltose-Binding Proteins
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Membrane Proteins
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Peptides
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Recombinant Fusion Proteins
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DNA
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Endopeptidases
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Dimyristoylphosphatidylcholine