Multicolor single-molecule FRET studies on dynamic protein systems

Abstract

Förster resonance energy transfer (FRET) is a powerful tool for studying protein conformations, interactions, and dynamics at the single-molecule level. Multicolor FRET extends conventional two-color FRET by incorporating three or more fluorophores and thereby enabling a more comprehensive view of complex biomolecular processes. This technique allows for the simultaneous tracking of multiple structural changes, detecting intermediate states, and resolving heterogeneous population distributions. In this review, we discuss the recent advancements in fluorophore labeling strategies and data analysis methods that have significantly improved the precision and applicability of multicolor FRET in protein studies. We then end this review by showcasing recent applications for investigating protein folding and processes involved in gene regulation.