Peptides in headlock--a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy

Scientific Reports
Michael B BraunUlrich Rothbauer

Abstract

Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies.

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Methods Mentioned

BETA
surface plasmon resonance
immunoprecipitation
transfection
phage display
pulldown
size exclusion chromatography
Protein Assay

Software Mentioned

refine
Phenix
COOT
PyMol
REFMAC5
XDS
ImageXpress
PHASER

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