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5‐Fluorotryptophan as dual probe for ground‐state heterogeneity and excited‐state dynamics in apoflavodoxin
Author(s) -
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.
Publication year - 2009
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2009.07.022
Subject(s) - azotobacter vinelandii , chemistry , tryptophan , fluorescence , anisotropy , excited state , förster resonance energy transfer , fluorescence anisotropy , crystallography , biochemistry , physics , amino acid , atomic physics , nitrogen , quantum mechanics , nitrogenase , organic chemistry , nitrogen fixation , membrane
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 19 F 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 19 F NMR and fluorescence spectroscopy.