title: A High-Throughput Fluorometric Assay for Lipid-Protein Binding.
abstract: An increasing number of intracellular and extracellular proteins are shown to interact with membrane lipids under physiological conditions. For rapid and robust quantitative measurement of lipid-protein interaction, we developed a sensitive fluorescence quenching-based assay that is universally applicable to all proteins and lipids. The assay employs fluorescence protein (FP)-tagged proteins whose fluorescence emission intensity is decreased when they bind vesicles containing quenching lipids. This simple assay can be performed with a fluorescence plate reader or a spectrofluorometer and optimized for different proteins with various combinations of FPs and quenching lipids. The assay allows a rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid-binding proteins, and high-throughput screening of molecules that modulate their membrane binding.
authors.affiliation: University of Illinois at Chicago, Chicago, IL, United States; Kyung Hee University, Yongin, South Korea. Electronic address: wcho@uic.edu.
authors.forename: W
authors.initials: W
authors.lastname: Cho
authors.affiliation: University of Illinois at Chicago, Chicago, IL, United States.
authors.forename: Y
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Kyung Hee University, Yongin, South Korea.
authors.forename: K
authors.initials: K
authors.lastname: Baek
authors.affiliation: University of Illinois at Chicago, Chicago, IL, United States.
authors.forename: H
authors.initials: H
authors.lastname: Kim
mesh_terms: D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
chemical_list: D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D018129:Phosphatidylinositol Phosphates; D011993:Recombinant Fusion Proteins; D053835:Unilamellar Liposomes; C485184:enhanced green fluorescent protein; C060974:phosphatidylinositol 3,4,5-triphosphate; D049452:Green Fluorescent Proteins; D004124:p-Dimethylaminoazobenzene; D005982:Glutathione Transferase; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt; C028694:1-palmitoyl-2-oleoylphosphatidylcholine
keywords: Dark quenchers; Fluorescence proteins; High-throughput fluorescence assay; Lipid specificity; Membrane protein binding; Membrane-binding inhibitors