A hot-spot motif characterizes the interface between a designed ankyrin-repeat protein and its target ligand

Luthur Siu Lun Cheung, Manu Kanwar, Marc Ostermeier, Konstantinos Konstantopoulos

Research output: Contribution to journalArticle

Abstract

Nonantibody scaffolds such as designed ankyrin repeat proteins (DARPins) can be rapidly engineered to detect diverse target proteins with high specificity and offer an attractive alternative to antibodies. Using molecular simulations, we predicted that the binding interface between DARPin off7 and its ligand (maltose binding protein; MBP) is characterized by a hot-spot motif in which binding energy is largely concentrated on a few amino acids. To experimentally test this prediction, we fused MBP to a transmembrane domain to properly orient the protein into a polymer-cushioned lipid bilayer, and characterized its interaction with off7 using force spectroscopy. Using this, to our knowledge, novel technique along with surface plasmon resonance, we validated the simulation predictions and characterized the effects of select mutations on the kinetics of the off7-MBP interaction. Our integrated approach offers scientific insights on how the engineered protein interacts with the target molecule.

Original languageEnglish (US)
Pages (from-to)407-416
Number of pages10
JournalBiophysical journal
Volume102
Issue number3
DOIs
StatePublished - Feb 8 2012

ASJC Scopus subject areas

  • Biophysics

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