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.

Keywords

• FRET
• MutS
• Msh2
• Msh6
• DNA mismatch repair
• SNARE proteins