title: Dynamics of light-induced activation in the PAS domain proteins LOV2 and PYP probed by time-resolved tryptophan fluorescence.
abstract: Light-induced activation of the LOV2-Jα domain of the photoreceptor phototropin from oat is believed to involve the detachment of the Jα helix from the central β-sheet and its subsequent unfolding. The dynamics of these conformational changes were monitored by time-resolved emission spectroscopy with 100 ns time resolution. Three transitions were detected during the LOV2-Jα photocycle with time constants of 3.4 μs, 500 μs, and 4.3 ms. The fastest transition is due to the decay of the flavin phosphorescence in the transition of the triplet LOV(L)(660) state to the singlet LOV(S)(390) signaling state. The 500 μs and 4.3 ms transitions are due to changes in tryptophan fluorescence and may be associated with the dissociation and unfolding of the Jα helix, respectively. They are absent in the transient absorption signal of the flavin chromophore. The tryptophan fluorescence signal monitors structural changes outside the chromophore binding pocket and indicates that there are at least three LOV(S)(390) intermediates. Since the 500 μs and 4.3 ms components are absent in a construct without the Jα helix and in the mutant W557S, the fluorescence signal is mainly due to tryptophan 557. The kinetics of the main 500 μs component is strongly temperature dependent with activation energy of 18.2 kcal/mol suggesting its association with a major structural change. In the structurally related PAS domain protein PYP the N-terminal cap dissociates from the central β-sheet and unfolds upon signaling state formation with a similar time constant of ∼1 ms. Using transient fluorescence we obtained a nearly identical activation energy of 18.5 kcal/mol for this transition.
authors.affiliation: Biophysics Group, Department of Physics, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Hoersch
authors.forename: Farzin
authors.initials: F
authors.lastname: Bolourchian
authors.forename: Harald
authors.initials: H
authors.lastname: Otto
authors.forename: Maarten P
authors.initials: MP
authors.lastname: Heyn
authors.forename: Roberto A
authors.initials: RA
authors.lastname: Bogomolni
mesh_terms: D018554:Avena / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D008027:Light; D008958:Models, Molecular; D056949:Phototropins / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
chemical_list: D056949:Phototropins; D014364:Tryptophan