title: Applying bimolecular fluorescence complementation to screen and purify aquaporin protein:protein complexes.
abstract: Protein:protein interactions play key functional roles in the molecular machinery of the cell. A major challenge for structural biology is to gain high-resolution structural insight into how membrane protein function is regulated by protein:protein interactions. To this end we present a method to express, detect, and purify stable membrane protein complexes that are suitable for further structural characterization. Our approach utilizes bimolecular fluorescence complementation (BiFC), whereby each protein of an interaction pair is fused to nonfluorescent fragments of yellow fluorescent protein (YFP) that combine and mature as the complex is formed. YFP thus facilitates the visualization of protein:protein interactions in vivo, stabilizes the assembled complex, and provides a fluorescent marker during purification. This technique is validated by observing the formation of stable homotetramers of human aquaporin 0 (AQP0). The method's broader applicability is demonstrated by visualizing the interactions of AQP0 and human aquaporin 1 (AQP1) with the cytoplasmic regulatory protein calmodulin (CaM). The dependence of the AQP0-CaM complex on the AQP0 C-terminus is also demonstrated since the C-terminal truncated construct provides a negative control. This screening approach may therefore facilitate the production and purification of membrane protein:protein complexes for later structural studies by X-ray crystallography or single particle electron microscopy.
authors.affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg, Göteborg, SE-405 30, Sweden.
authors.forename: Jennie
authors.initials: J
authors.lastname: Sjöhamn
authors.affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg, Göteborg, SE-405 30, Sweden.
authors.forename: Petra
authors.initials: P
authors.lastname: Båth
authors.affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg, Göteborg, SE-405 30, Sweden.
authors.forename: Richard
authors.initials: R
authors.lastname: Neutze
authors.affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg, Göteborg, SE-405 30, Sweden.
authors.forename: Kristina
authors.initials: K
authors.lastname: Hedfalk
mesh_terms: D051398:Aquaporin 1 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020346:Aquaporins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002147:Calmodulin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005136:Eye Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005816:Genetic Complementation Test; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: C496020:AQP1 protein, human; D020346:Aquaporins; D001426:Bacterial Proteins; D002147:Calmodulin; D005136:Eye Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C030328:aquaporin 0; C054562:yellow fluorescent protein, Bacteria; D051398:Aquaporin 1
keywords: BiFC; Saccharomyces cerevisiae; YFP; aquaporin; calmodulin; membrane protein complex; protein-protein interaction