Probing Protein Conformations by in Situ Non-Covalent Fluorescence Labeling Export

@article{citeulike:4010147, abstract = {doi: 10.1021/bc8002088 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 tris-NTA/fluorophore conjugates, which bind histidine-tagged proteins with high affinity, for site-specific 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.}, author = {Strunk, Jennifer J. and Gregor, Ingo and Becker, Yvonne and Lamken, Peter and Lata, Suman and Reichel, Annett and Enderlein, J\"{o}rg and Piehler, Jacob}, citeulike-article-id = {4010147}, citeulike-linkout-0 = {http://dx.doi.org/10.1021/bc8002088}, citeulike-linkout-1 = {http://pubs.acs.org/doi/abs/10.1021/bc8002088}, day = {21}, doi = {10.1021/bc8002088}, journal = {Bioconjugate Chemistry}, keywords = {fluorescent-tag, fluorophore, protein-dynamics}, month = {January}, number = {1}, pages = {41--46}, posted-at = {2009-02-05 19:00:42}, priority = {2}, title = {Probing Protein Conformations by in Situ Non-Covalent Fluorescence Labeling}, url = {http://dx.doi.org/10.1021/bc8002088}, volume = {20}, year = {2009} }

TY - JOUR ID - citeulike:4010147 L3 - citeulike-article-id:4010147 N2 - doi: 10.1021/bc8002088 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 tris-NTA/fluorophore conjugates, which bind histidine-tagged proteins with high affinity, for site-specific 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. IS - 1 JF - Bioconjugate Chemistry EP - 46 TI - Probing Protein Conformations by in Situ Non-Covalent Fluorescence Labeling VL - 20 SP - 41 KW - fluorescent-tag KW - fluorophore KW - protein-dynamics AU - Strunk, Jennifer AU - Gregor, Ingo AU - Becker, Yvonne AU - Lamken, Peter AU - Lata, Suman AU - Reichel, Annett AU - Enderlein, Jörg AU - Piehler, Jacob PY - 2009/01/21/ UR - http://dx.doi.org/10.1021/bc8002088 ER -