Thioamides as fluorescence quenching probes: minimalist chromophores to monitor protein dynamics.
Decreasing the size of spectroscopic probes can afford higher-resolution structural information from fluorescence experiments. Therefore, we have developed p-cyanophenylalanine (Cnf) and backbone thioamides as a fluorophore/quencher pair. Through the examination of a series of thiopeptides, we have determined the working distance for this pair to be 8-30 Å. We have also carried out a proof-of-principle protein-folding experiment in which a Cnf/thioamide-labeled version of villin headpiece HP35 was thermally unfolded while the Cnf/thioamide distance was monitored by fluorescence. For a given protein, thioamide substitutions could be used to track motions with a much greater number of measurements than for current fluorescence probes, providing a dense array of data with which to model conformational changes.
Goldberg
Jacob M
JM
Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, United States.
Batjargal
Solongo
S
Petersson
E James
EJ
D005456:Fluorescent Dyes / Q000737:chemistry*; D008958:Models, Molecular; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013854:Thioamides / Q000737:chemistry*
D005456:Fluorescent Dyes; D011506:Proteins; D013854:Thioamides