Probing protein conformations by in situ non-covalent flourescence labeling

JJ Strunk, I Gregor, Y Becker, P Lamken, S Lata, A Reichel, J Enderlein, J Piehler

Research output: Contribution to journalArticle (Academic Journal)peer-review

19 Citations (Scopus)


The conformational dynamics of proteins plays a key role in their complex physiological functions. Fluorescence resonance energy transfer (FRET) is a particular powerful tool for studying protein conformational dynamics, but requires efficient site-specific labeling with fluorescent reporter probes. We have employed different Iris-NTA/fluorophore conjugates, which bind histidine-tagged proteins with high affinity, for sitespecific incorporation of FRET acceptors into proteins, which were covalently labeled with a donor fluorophore. We demonstrate versatile application of this approach for exploring the conformation of the type I interferon receptor ectodomains ifnar1-EC and ifnar2-EC. Substantial ligand-induced conformational changes of ifnar1-EC, but not ifnar2-EC, were observed by monitoring the fluorescence intensity and the fluorescence lifetime of the FRET donor. Time-resolved fluorescence correlation spectroscopy revealed a substantial conformational flexibility of ifnar1-EC and a ligand-induced tightening. Our results demonstrate that protein labeling with tris-NTA/fluorophores enables for efficient quantitative intramolecular FRET analysis.
Translated title of the contributionProbing protein conformations by in situ non-covalent flourescence labeling
Original languageEnglish
Pages (from-to)41 - 46
Number of pages6
JournalBioconjugate Chemistry
Volume20 (1)
Publication statusPublished - Jan 2009


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