title: Analysis of Protein-Lipid Interactions Using Purified C2 Domains.
abstract: C2 domains (C2s) are regulatory protein modules identified in eukaryotic proteins targeted to cell membranes. C2s were initially characterized as independently folded Ca(2+)-dependent phospholipids binding domains; however, later studies have shown that C2s have evolutionarily diverged into Ca(2+)-dependent and Ca(2+)-independent forms. These forms interact and regulate their affinity to diverse lipid species using different binding mechanisms. In this protocol we describe a biochemical approach to produce, purify, and solubilize functional C2 domains bound to GST for the identification of their putative Ca(2+)-dependent and Ca(2+)-independent lipid-binding partners.
authors.affiliation: Departamento de Biología Molecular y Bioquímica, Instituto de Hortofruticultura Subtropical y Mediterránea (IHSM-UMA-CSIC), Málaga, Spain.
authors.forename: Jessica
authors.initials: J
authors.lastname: Pérez-Sancho
authors.affiliation: Center for Research in Agricultural Genomics CRAG (CSIC-IRTA-UAB-UB), Edifici CRAG-Campus UAB, Bellaterra (Cerdanyola del Vallés), Barcelona, 08193, Spain.
authors.forename: Arnaldo L
authors.initials: AL
authors.lastname: Schapire
authors.affiliation: Departamento de Biología Molecular y Bioquímica, Instituto de Hortofruticultura Subtropical y Mediterránea (IHSM-UMA-CSIC), Málaga, Spain.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Botella
authors.affiliation: Department of Botany, Faculty of Sciences, University of British Columbia, 6270 University Blvd.,, Vancouver, BC, Canada, V6T 1Z4. abel.rosado@botany.ubc.ca.
authors.forename: Abel
authors.initials: A
authors.lastname: Rosado
mesh_terms: D002118:Calcium / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D008563:Membrane Lipids; D010743:Phospholipids; D011506:Proteins; D011993:Recombinant Fusion Proteins; D002118:Calcium
keywords: C2 domain; Ca2+-dependent lipid binding; Multilamellar vesicles; Protein-lipid overlay assay; Solubility