title: Unfolding of the C-Terminal Jα Helix in the LOV2 Photoreceptor Domain Observed by Time-Resolved Vibrational Spectroscopy.
abstract: Light-triggered reactions of biological photoreceptors have gained immense attention for their role as molecular switches in their native organisms and for optogenetic application. The light, oxygen, and voltage 2 (LOV2) sensing domain of plant phototropin binds a C-terminal Jα helix that is docked on a β-sheet and unfolds upon light absorption by the flavin mononucleotide (FMN) chromophore. In this work, the signal transduction pathway of LOV2 from Avena sativa was investigated using time-resolved infrared spectroscopy from picoseconds to microseconds. In D2O buffer, FMN singlet-to-triplet conversion occurs in 2 ns and formation of the covalent cysteinyl-FMN adduct in 10 μs. We observe a two-step unfolding of the Jα helix: The first phase occurs concomitantly with Cys-FMN covalent adduct formation in 10 μs, along with hydrogen-bond rupture of the FMN C4═O with Gln-513, motion of the β-sheet, and an additional helical element. The second phase occurs in approximately 240 μs. The final spectrum at 500 μs is essentially identical to the steady-state light-minus-dark Fourier transform infrared spectrum, indicating that Jα helix unfolding is complete on that time scale.
authors.affiliation: Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit , 1081 De Boelelaan, 1081HV Amsterdam, The Netherlands.
authors.forename: Patrick E
authors.initials: PE
authors.lastname: Konold
authors.affiliation: Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit , 1081 De Boelelaan, 1081HV Amsterdam, The Netherlands.
authors.forename: Tilo
authors.initials: T
authors.lastname: Mathes
authors.affiliation: Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit , 1081 De Boelelaan, 1081HV Amsterdam, The Netherlands.
authors.forename: Jörn
authors.initials: J
authors.lastname: Weiβenborn
authors.affiliation: Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit , 1081 De Boelelaan, 1081HV Amsterdam, The Netherlands.
authors.forename: Marie Louise
authors.initials: ML
authors.lastname: Groot
authors.affiliation: Department of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin , Invalidenstraße 42, 10115 Berlin, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Hegemann
authors.affiliation: Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit , 1081 De Boelelaan, 1081HV Amsterdam, The Netherlands.
authors.forename: John T M
authors.initials: JT
authors.lastname: Kennis
mesh_terms: D029681:Arabidopsis Proteins / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D020283:Photoreceptors, Microbial / Q000737:chemistry*; D058767:Protein Unfolding; D013057:Spectrum Analysis / Q000379:methods*; D014732:Vibration
chemical_list: D029681:Arabidopsis Proteins; D004268:DNA-Binding Proteins; C000612094:LOV2 protein, Arabidopsis; D020283:Photoreceptors, Microbial