A novel flow cytometric assay to quantify interactions between proteins and membrane lipids.
A diverse set of experimental systems has been developed to probe protein-lipid interactions. These include measurements with the headgroups of membrane lipids in solution, immobilized membrane lipids, and analysis of protein binding to membrane lipids reconstituted in liposomes. Each of these methodologies has strengths but also substantial limitations. For example, measurements between proteins and lipid headgroups or with immobilized membrane lipids do not probe interactions in their natural environment, the lipid bilayer. The use of liposomes, however, was so far mostly restricted to biochemical flotation experiments that do not provide quantitative and/or kinetic data. Here, we present a fast and sensitive flow cytometric method to detect protein-lipid interactions. This technique allows for quantitative measurements of interactions between multiple fluorescently labeled proteins and membrane lipids reconstituted in lipid bilayers. The assay can be used to quantify binding efficiencies and to determine kinetic constants. The method is further characterized by a short sampling time of only a few seconds that allows for high-content screening procedures. Finally, using light scatter measurements, the described method also allows for monitoring changes of membrane curvature as well as tethering of liposomes evoked by binding of proteins.
Temmerman
Koen
K
Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany.
Nickel
Walter
W
D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods* / Q000706:statistics & numerical data; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D008027:Light; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism*; D010316:Particle Size; D054803:Phospholipase C delta / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012542:Scattering, Radiation; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008081:Liposomes; D008563:Membrane Lipids; D011506:Proteins; D011993:Recombinant Fusion Proteins; D016222:Fibroblast Growth Factor 2; D049452:Green Fluorescent Proteins; D054803:Phospholipase C delta