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Proceedings Paper

Low-temperature and time-resolved spectroscopic characterization of the LOV2 domain of Avena sativa phototropin 1
Author(s): Magdalena Gauden; Sean Crosson; I. H. M. van Stokkum; Rienk van Grondelle; Keith Moffat; John T. M. Kennis
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Paper Abstract

The phototropins are plant blue-light receptors that base their light-dependent action on the reversible formation of a covalent bond between a flavin mononucleotide (FMN) cofactor and a conserved cysteine residue in light, oxygen or voltage (LOV) domains. The spectroscopic properties of the LOV2 domain of phototropin 1 of Avena sativa (oat) have been investigated by means of low-temperature absorption and fluorescence spectroscopy and by time-resolved fluorescence spectroscopy. The low-temperature absorption spectrum of the LOV2 domain showed a fine structure around 473 nm, indicating heterogeneity in the flavin binding pocket. The fluorescence quantum yield of the flavin cofactor increased from 0.13 to 0.41 upon cooling the sample from room temperature to 77 K. A pronounced phosphorescence emission around 600 nm was observed in the LOV2 domain between 77 and 120 K, allowing for an accurate positioning of the flavin triplet state in the LOV2 domain at 16900 cm-1. Fluorescence from the cryotrapped covalent adduct state was extremely weak, with a fluorescence spectrum showing a maximum at 440 nm. Time-resolved fluorescence experiments utilizing a synchroscan streak camera revealed a singlet-excited state lifetime of the LOV2 domain of 2.4 ns. FMN dissolved in aqueous solution showed a pH-dependent lifetime ranging between 2.9 ns at pH 2.0 to 4.7 ns at pH 8.0. No spectral shifting of the flavin emission was observed in the LOV2 domain nor in FMN in aqueous solution.

Paper Details

Date Published: 8 September 2004
PDF: 8 pages
Proc. SPIE 5463, Femtosecond Laser Applications in Biology, (8 September 2004); doi: 10.1117/12.548054
Show Author Affiliations
Magdalena Gauden, Vrije Univ. Amsterdam (Netherlands)
Sean Crosson, Univ. of Chicago (United States)
I. H. M. van Stokkum, Vrije Univ. Amsterdam (Netherlands)
Rienk van Grondelle, Vrije Univ. Amsterdam (Netherlands)
Keith Moffat, Univ. of Chicago (United States)
John T. M. Kennis, Vrije Univ. Amsterdam (Netherlands)

Published in SPIE Proceedings Vol. 5463:
Femtosecond Laser Applications in Biology
Sigrid Avrillier; Jean-Michel Tualle, Editor(s)

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