title: Minimalist probes for studying protein dynamics: thioamide quenching of selectively excitable fluorescent amino acids.
abstract: Fluorescent probe pairs that can be selectively excited in the presence of Trp and Tyr are of great utility in studying conformational changes in proteins. However, the size of these probe pairs can restrict their incorporation to small portions of a protein sequence where their effects on secondary and tertiary structure can be tolerated. Our findings show that a thioamide bond-a single atom substitution of the peptide backbone-can quench fluorophores that are red-shifted from intrinsic protein fluorescence, such as acridone. Using steady-state and fluorescence lifetime measurements, we further demonstrate that this quenching occurs through a dynamic electron-transfer mechanism. In a proof-of-principle experiment, we apply this technique to monitor unfolding in a model peptide system, the villin headpiece HP35 fragment. Thioamide analogues of the natural amino acids can be placed in a variety of locations in a protein sequence, allowing one to make a large number of measurements to model protein folding.
authors.affiliation: Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, USA.
authors.forename: Jacob M
authors.initials: JM
authors.lastname: Goldberg
authors.forename: Lee C
authors.initials: LC
authors.lastname: Speight
authors.forename: Mark W
authors.initials: MW
authors.lastname: Fegley
authors.forename: E James
authors.initials: EJ
authors.lastname: Petersson
mesh_terms: D000166:Acridines / Q000737:chemistry; D054831:Acridones; D000596:Amino Acids / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D015345:Oligonucleotide Probes / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013854:Thioamides / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000166:Acridines; D054831:Acridones; D000596:Amino Acids; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D010455:Peptides; D011506:Proteins; D013854:Thioamides; D014443:Tyrosine; C041300:acridone; D014364:Tryptophan