title: High-throughput fluorescence assay for membrane-protein interaction.
abstract: Membrane-protein interaction plays key roles in a wide variety of biological processes. Although various methods have been employed to measure membrane binding of soluble proteins, a robust high-throughput assay that is universally applicable to all proteins is lacking at present. Here we report a new fluorescence quenching assay utilizing enhanced green fluorescence protein (EGFP)-fusion proteins and a lipid containing a dark quencher, N-dimethylaminoazobenzenesulfonyl-phosphatidylethanolamine (dabsyl-PE). The EGFP fluorescence emission intensity showed a large decrease (i.e., >50%) when EGFP-fusion proteins bound the vesicles containing 5 mol% dabsyl-PE. This simple assay, which can be performed using either a cuvette-based spectrofluorometer or a fluorescence plate reader, allowed rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid binding domains, including two pleckstrin homology domains, an epsin N-terminal homology domain, and a phox homology domain. The assay can also be applied to high-throughput screening of small molecules that modulate membrane binding of proteins.
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607.
authors.forename: Hyunjin
authors.initials: H
authors.lastname: Kim
authors.forename: Hamid Samareh
authors.initials: HS
authors.lastname: Afsari
authors.forename: Wonhwa
authors.initials: W
authors.lastname: Cho
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry; D011485:Protein Binding / Q000187:drug effects; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D008563:Membrane Lipids; D010714:Phosphatidylethanolamines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C483858:phosphatidylethanolamine
keywords: dark quencher; green fluorescence protein; high-throughput assay; membrane-protein binding