Journal
JOURNAL OF LIPID RESEARCH
Volume 54, Issue 12, Pages 3531-3538Publisher
ELSEVIER
DOI: 10.1194/jlr.D041376
Keywords
membrane-protein binding; high-throughput assay; dark quencher; green fluorescence protein
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Funding
- National Institutes of Health [GM-68849, GM-52598]
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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.jlr The assay can also be applied to high-throughput screening of small molecules that modulate membrane binding of proteins.
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