title: High-throughput protein structural analysis using site-directed fluorescence labeling and the bimane derivative (2-pyridyl)dithiobimane.
abstract: We present a site-directed fluorescence labeling (SDFL) study of 25 different T4 lysozyme protein samples labeled with the thiol-cleavable fluorophore, (2-pyridyl)dithiobimane (PDT-Bimane). Our results demonstrate PDT-Bimane can be used in cysteine-scanning studies to detect protein secondary structure, and to map proximity between sites in proteins by monitoring tryptophan quenching of bimane fluorescence. In addition, the reducible nature of PDT-Bimane can be exploited to resolve problems often faced in SDFL studies: ensuring specific labeling of cysteine residues, determining the extent of free label contamination, and accurately determining labeling efficiency even at low concentrations. The ability to cleave PDT-Bimane off the protein enables rapid determination of these parameters, and positions it as an ideal fluorophore for automated, high-throughput structural studies of protein folding, the detection of protein-protein interactions, and the monitoring of real-time conformational changes.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239-3098, USA.
authors.forename: Steven E
authors.initials: SE
authors.lastname: Mansoor
authors.forename: David L
authors.initials: DL
authors.lastname: Farrens
mesh_terms: D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D012997:Solvents / Q000737:chemistry
chemical_list: C490156:(2-pyridyl)dithiobimane; D019086:Bridged Bicyclo Compounds, Heterocyclic; D004220:Disulfides; D005456:Fluorescent Dyes; D012997:Solvents