Time-resolved Fourier transform infrared study on photoadduct formation and secondary structural changes within the phototropin LOV domain.
Phototropins are plant blue-light photoreceptors containing two light-, oxygen-, or voltage-sensitive (LOV) domains and a C-terminal kinase domain. The two LOV domains bind noncovalently flavin mononucleotide as a chromophore. We investigated the photocycle of fast-recovery mutant LOV2-I403V from Arabidopsis phototropin 2 by step-scan Fourier transform infrared spectroscopy. The reaction of the triplet excited state of flavin with cysteine takes place with a time constant of 3 micros to yield the covalent adduct. Our data provide evidence that the flavin is unprotonated in the productive triplet state, disfavoring an ionic mechanism of bond formation. An intermediate adduct species was evident that displayed changes in secondary structure in the helix or loop region, and relaxed with a time constant of 120 micros. In milliseconds, the final adduct state is formed by further alterations of secondary structure, including beta-sheets. A comparison with wild-type adduct spectra shows that the mutation does not interfere with the functionality of the domain. All signals originate from within the LOV domain, because the construct does not comprise the adjacent Jalpha helix required for signal transduction. The contribution of early and late adduct intermediates to signal transfer to the Jalpha helix outside of the domain is discussed.
Pfeifer
Anna
A
Biophysical Chemistry, Department of Chemistry, Bielefeld University, 33615 Bielefeld, Germany.
Majerus
Teresa
T
Zikihara
Kazunori
K
Matsuoka
Daisuke
D
Tokutomi
Satoru
S
Heberle
Joachim
J
Kottke
Tilman
T
D017360:Arabidopsis / Q000737:chemistry; D029681:Arabidopsis Proteins; D056931:Cryptochromes; D003545:Cysteine / Q000737:chemistry; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D007834:Lasers; D008027:Light; D008958:Models, Molecular; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared
D029681:Arabidopsis Proteins; C538962:CRY1 protein, Arabidopsis; D056931:Cryptochromes; D005420:Flavoproteins; D003545:Cysteine