title: Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes.
abstract: To enable studies of conformational changes within multimolecular complexes, we present a simultaneous, four-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera-based, wide-field detection. We further demonstrate labeling histidine-tagged proteins noncovalently with Tris-nitrilotriacetic acid (Tris-NTA)-conjugated dyes to achieve single molecule detection. We combine these methods to colocalize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes.
authors.affiliation: Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. derie@unc.edu
authors.forename: Vanessa
authors.initials: V
authors.lastname: DeRocco
authors.forename: Trevor
authors.initials: T
authors.lastname: Anderson
authors.forename: Jacob
authors.initials: J
authors.lastname: Piehler
authors.forename: Dorothy A
authors.initials: DA
authors.lastname: Erie
authors.forename: Keith
authors.initials: K
authors.lastname: Weninger
mesh_terms: D004396:Coloring Agents; D004247:DNA / Q000737:chemistry; D053843:DNA Mismatch Repair; D004735:Energy Transfer; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006639:Histidine; D046911:Macromolecular Substances / Q000737:chemistry*; D051719:MutS DNA Mismatch-Binding Protein / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D009571:Nitrilotriacetic Acid; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D011487:Protein Conformation / Q000187:drug effects; D050600:SNARE Proteins / Q000737:chemistry; D014325:Tromethamine
chemical_list: D004396:Coloring Agents; D046911:Macromolecular Substances; D050600:SNARE Proteins; D014325:Tromethamine; D006639:Histidine; D004247:DNA; D051719:MutS DNA Mismatch-Binding Protein; D009571:Nitrilotriacetic Acid