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 MutS alpha. on DNA while monitoring MutS alpha-induced DNA bending using Forster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes.