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5-Fluorotryptophan as dual probe for ground-state heterogeneity and excited-state dynamics in apoflavodoxin |
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| Authors: | N.V. Visser S.M. Nabuurs C.P.M. van Mierlo J. Broos |
| Affiliation: | a Laboratory of Biophysics, Microspectroscopy Centre, Wageningen University, P.O. Box 8128, 6700 ET Wageningen, The Netherlands b Laboratory of Biochemistry, Microspectroscopy Centre, Wageningen University, P.O. Box 8128, 6700 ET Wageningen, The Netherlands c Department of Physics, University of Strathclyde, Scottish Universities Physics Alliance, Photophysics Group, Glasgow G4 0NG, United Kingdom d Department of Biophysical Chemistry and Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands |
| Abstract: | The apoflavodoxin protein from Azotobacter vinelandii harboring three tryptophan (Trp) residues, was biosynthetically labeled with 5-fluorotryptophan (5-FTrp). 5-FTrp has the advantage that chemical differences in its microenvironment can be sensitively visualized via 19F NMR. Moreover, it shows simpler fluorescence decay kinetics. The occurrence of FRET was earlier observed via the fluorescence anisotropy decay of WT apoflavodoxin and the anisotropy decay parameters are in excellent agreement with distances between and relative orientations of all Trp residues. The anisotropy decay in 5-FTrp apoflavodoxin demonstrates that the distances and orientations are identical for this protein. This work demonstrates the added value of replacing Trp by 5-FTrp to study structural features of proteins via 19F NMR and fluorescence spectroscopy. |
| Keywords: | Fluorescence anisotropy 19F NMR Picosecond fluorescence Protein folding Protein stability |
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