title: An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities.
abstract: Identifying and characterizing protein interactions are fundamental steps toward understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the colocalization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecular tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin alpha (Imp alpha), and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified colocalization assay is sensitive enough to detect protein interactions with K(d) values that span over four orders of magnitude (1 nM to 15 microM). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris.
authors.affiliation: University of Tennessee-Oak Ridge National Laboratory, Graduate School of Genome Science and Technology, Knoxville, TN 37996, USA.
authors.forename: A Nicole
authors.initials: AN
authors.lastname: Edwards
authors.forename: Jason D
authors.initials: JD
authors.lastname: Fowlkes
authors.forename: Elizabeth T
authors.initials: ET
authors.lastname: Owens
authors.forename: Robert F
authors.initials: RF
authors.lastname: Standaert
authors.forename: Dale A
authors.initials: DA
authors.lastname: Pelletier
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Hurst
authors.forename: Mitchel J
authors.initials: MJ
authors.lastname: Doktycz
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Morrell-Falvey
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis; D018797:Cell Cycle Proteins / Q000032:analysis; D049452:Green Fluorescent Proteins / Q000032:analysis; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000032:analysis; D010957:Plasmids / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis*; D012241:Rhodopseudomonas / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D028901:alpha Karyopherins / Q000032:analysis
chemical_list: D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; C104636:DivIVA protein, bacteria; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins; D049452:Green Fluorescent Proteins