Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful tool to probe macromolecular dynamics. Two-color smFRET, however, provides only one-dimensional distance information, limiting its use in complex systems. Multicolor smFRET with three or more fluorophores can overcome this, though three is often the practical limit due to the narrow selection of dyes with suitable spectral properties, the challenges of labeling with multiple fluorophores, and the complexity of data interpretation. Here, we survey recent investigations of the dynamics of macromolecules and their complexes based on three-color smFRET. In this review, three-color smFRET refers to the use of three fluorophores in a FRET system where two or all three fluorophores may participate in FRET at a given time point. We aim to highlight structural biology problems particularly well suited for three-color smFRET and to provide a brief overview of designing and implementing such experiments in light of their benefits and challenges.
Gao et al. (Wed,) studied this question.