title: A Quantitative Fluorescence-Based Assay for Assessing LIM Domain-Peptide Interactions.
abstract: We have developed Förster resonance energy transfer (FRET)-based experiments for measuring the binding affinity, off-rates, and inferred on-rates for interactions between a family of transcriptional regulators and their intrinsically disordered binding partners. It was difficult to evaluate these interactions previously, as the transcriptional regulators are obligate binding proteins that aggregate in the absence of a binding partner. The assays rely on fusion constructs where binding domains are linked by a flexible tether containing a specific protease site, with fluorescent proteins at either end that display FRET when the complex is formed. Loss of FRET is monitored after cutting the tether followed by dilution or competition with a non-fluorescent peptide. These methods allowed a wide range of binding affinities (10-9-10-5m) to be determined. Our data indicate that interactions of closely related proteins can have surprisingly different binding properties.
authors.affiliation: School of Life and Environmental Sciences, The University of Sydney, NSW, 2006, Australia.
authors.forename: Neil O
authors.initials: NO
authors.lastname: Robertson
authors.affiliation: School of Life and Environmental Sciences, The University of Sydney, NSW, 2006, Australia.
authors.forename: Manan
authors.initials: M
authors.lastname: Shah
authors.affiliation: School of Life and Environmental Sciences, The University of Sydney, NSW, 2006, Australia. jacqui.matthews@sydney.edu.au.
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Matthews
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D060588:LIM Domain Proteins / Q000737:chemistry*; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*
chemical_list: D060588:LIM Domain Proteins; D010455:Peptides
keywords: FRET; fluorescence; kinetics; protein engineering; protein-protein interactions