OS-FRET: a new one-sample method for improved FRET measurements.
Fluorescence resonance energy transfer (FRET) is a powerful tool for studying macromolecular assemblies in vitro under near-physiological conditions. Here we present a new type of one-sample FRET (OS-FRET) method employing a novel, nonfluorescent methanethiosulfonate-linked acceptor that can be reversibly coupled to a target sulfhydryl residue via a disulfide bond. After the quenched donor emission is quantitated, the acceptor is removed by reduction, allowing measurement of unquenched donor emission in the same sample. Previous one-sample methods provide distinct advantages in specific FRET applications. The new OS-FRET method is a generalizable spectrochemical approach that can be applied to macromolecular systems lacking essential disulfide bonds and eliminates the potential systematic errors of some earlier one-sample methods. In addition, OS-FRET enables quantitative FRET measurements in virtually any fluorescence spectrometer or detection device. Compared to conventional multisample FRET methods, OS-FRET conserves sample, increases the precision of data, and shortens the time per measurement. The utility of the method is illustrated by its application to a protein complex of known structure formed by CheW and the P4-P5 fragment of CheA, both from Thermotoga maritima. The findings confirm the practicality and advantages of OS-FRET. Anticipated applications of OS-FRET include analysis of macromolecular structure, binding and conformational dynamics, and high-throughput screening for interactions and inhibitors.
Department of Chemistry and Biochemistry and Molecular Biophysics Program, University of Colorado, Boulder, CO 80309-0215, USA.
Annette H
AH
Erbse
Adam J
AJ
Berlinberg
Ching-Ying
CY
Cheung
Wai-Yee
WY
Leung
Joseph J
JJ
Falke
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D002633:Chemotaxis / Q000235:genetics; D018360:Crystallography, X-Ray; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods* / Q000592:standards; D046911:Macromolecular Substances / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D000072236:Methyl-Accepting Chemotaxis Proteins; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods / Q000592:standards; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D010446:Peptide Fragments; C051236:CheW protein, Bacteria