Many proteins are dynamic and can adopt a broad range of conformations in solution. Confocal single-molecule FRET experiments, performed on the EI-FLEX, provide a straightforward method to explore the full range for conformations adopted by proteins under physiologically relevant conditions.
The experiment measures the FRET efficiency between two fluorescent labels, on a molecule by molecule basis. It is simple to implement and enables the investigation of protein conformational changes upon the binding of different ligands, for example, the interaction of nucleotides with DNA polymerase as shown in Fig. 1.
Experiments such as these, which help reveal the full range of conformations adopted by proteins, can give an insight into correct ligand binding and the existence of cryptic binding sites.
Fig 1. Example of the conformational landscapes of DNA polymerase in the presence of different nucleotides obtained from single-molecule FRET in solution.
References
Hohlbein, J., Aigrain, L., Craggs, T. et al. Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion. Nat Commun 4, 2131 (2013). https://doi.org/10.1038/ncomms3131