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.
Publication types
-
Research Support, N.I.H., Extramural
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Coloring Agents
-
DNA / chemistry
-
DNA Mismatch Repair
-
Energy Transfer
-
Fluorescence Resonance Energy Transfer / methods*
-
Histidine
-
Macromolecular Substances / chemistry*
-
MutS DNA Mismatch-Binding Protein / analysis
-
MutS DNA Mismatch-Binding Protein / chemistry
-
MutS DNA Mismatch-Binding Protein / pharmacology
-
Nitrilotriacetic Acid
-
Nucleic Acid Conformation / drug effects*
-
Protein Conformation / drug effects
-
SNARE Proteins / chemistry
-
Tromethamine
Substances
-
Coloring Agents
-
Macromolecular Substances
-
SNARE Proteins
-
Tromethamine
-
Histidine
-
DNA
-
MutS DNA Mismatch-Binding Protein
-
Nitrilotriacetic Acid