Proper protein function in cells, tissues and organisms depends critically on correct protein folding or interaction with partners. Over the last decade, single-molecule FRET (smFRET) has emerged as a powerful tool to probe complex distributions, dynamics, pathways and landscapes in protein folding and binding reactions, leveraging its ability to avoid averaging over an ensemble of molecules. While smFRET was practiced in a two-color form until recently, the last few years have seen the development of enhanced multicolor smFRET methods that provide additional structural information permitting us to probe more complex mechanisms. In this review, we provide a brief introduction to the smFRET technique, then follow with advanced multicolor measurements and end with ongoing methodology developments in microfluidics and protein labeling that are beginning to make these techniques more broadly applicable to answering a number of key questions about folding and binding.
