5-Fluorotryptophan as dual probe for ground-state heterogeneity and excited-state dynamics in apoflavodoxin

Visser, N.V.; Westphal, A.H.; Nabuurs, S.M.; van Hoek, A.; van Mierlo, C.P.M.; Visser, A.J.W.G.; Broos, J.; van Amerongen, H.

doi:10.1016/j.febslet.2009.07.022

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Volume 583, Issue 17 , Pages 2785-2788, 3 September 2009

5-Fluorotryptophan as dual probe for ground-state heterogeneity and excited-state dynamics in apoflavodoxin

Edited by Richard Cogdell

N.V. Visser
- Laboratory of Biophysics, Microspectroscopy Centre, Wageningen University, P.O. Box 8128, 6700 ET Wageningen, The Netherlands
A.H. Westphal
- Laboratory of Biochemistry, Microspectroscopy Centre, Wageningen University, P.O. Box 8128, 6700 ET Wageningen, The Netherlands
S.M. Nabuurs
- Laboratory of Biochemistry, Microspectroscopy Centre, Wageningen University, P.O. Box 8128, 6700 ET Wageningen, The Netherlands
A. van Hoek
- Laboratory of Biophysics, Microspectroscopy Centre, Wageningen University, P.O. Box 8128, 6700 ET Wageningen, The Netherlands
C.P.M. van Mierlo
- Laboratory of Biochemistry, Microspectroscopy Centre, Wageningen University, P.O. Box 8128, 6700 ET Wageningen, The Netherlands
A.J.W.G. Visser
- Department of Physics, University of Strathclyde, Scottish Universities Physics Alliance, Photophysics Group, Glasgow G4 0NG, United Kingdom
J. Broos
- Department of Biophysical Chemistry and Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
H. van Amerongen
- Laboratory of Biophysics, Microspectroscopy Centre, Wageningen University, P.O. Box 8128, 6700 ET Wageningen, The Netherlands
- Corresponding author. Fax: +31 317 482725.

Received 16 June 2009; received in revised form 13 July 2009; accepted 13 July 2009. published online 20 July 2009.

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