High-throughput protein structural analysis using site-directed fluorescence labeling and the bimane derivative (2-pyridyl)dithiobimane

Steven E. Mansoor, David Farrens

Research output: Contribution to journalArticle

44 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)9426-9438
Number of pages13
JournalBiochemistry
Volume43
Issue number29
DOIs
StatePublished - Jul 27 2004

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Structural analysis
Labeling
Fluorescence
Throughput
Derivatives
Proteins
Fluorophores
Cysteine
Secondary Protein Structure
Protein Folding
Protein folding
Muramidase
Sulfhydryl Compounds
Monitoring
Tryptophan
bimanes
(2-pyridyl)dithiobimane
Labels
Quenching
Contamination

ASJC Scopus subject areas

  • Biochemistry

Cite this

High-throughput protein structural analysis using site-directed fluorescence labeling and the bimane derivative (2-pyridyl)dithiobimane. / Mansoor, Steven E.; Farrens, David.

In: Biochemistry, Vol. 43, No. 29, 27.07.2004, p. 9426-9438.

Research output: Contribution to journalArticle

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