Allosterically Regulated Unfolding of the A′α Helix Exposes the Dimerization Site of the Blue-Light-Sensing Aureochrome-LOV Domain

Aureochromes have been shown to act as blue-light-regulated transcription factors in algae in the absence of phototropins. Aureochromes comprise a light-, oxygen-, or voltage-sensitive (LOV) domain as a sensory module binding the flavin chromophore and a basic region leucine zipper (bZIP) domain as an effector. The domain arrangement in aureochromes with an N-terminal effector is inversed to other LOV proteins. To clarify the role of the linking A′α helix in signaling, we have investigated the LOV domain of aureochrome1a from the diatom alga <i>Phaeodactylum tricornutum</i> without the N-terminal A′α helix but with the C-terminal Jα helix. Results were analyzed in comparison to those previously obtained on the LOV domain with both flanking helices and on the LOV domain with the A′α helix but without the Jα helix. Fourier transform infrared difference spectroscopy provides evidence by a band at 1656 cm^–1 that the A′α helix unfolds in response to light. This unfolding takes place only in the presence and as a consequence of the unfolding of the Jα helix, which points to an allosteric regulation. Size exclusion chromatography shows the LOV domain to be dimeric in the absence and monomeric in the presence of the A′α helix, implying that the folded helix covers the dimerization site. Therefore, the A′α helix directly modulates the oligomerization state of the LOV domain, whereas the Jα helix acts as an allosteric regulator. Both the allosteric control and the light-induced dimerization have not been observed in phototropin-LOV2 and point to a different signaling mechanism within the full-length proteins

Microsecond Deprotonation of Aspartic Acid and Response of the α/β Subdomain Precede C‑Terminal Signaling in the Blue Light Sensor Plant Cryptochrome

Plant cryptochromes are photosensory receptors that regulate various central aspects of plant growth and development. These receptors consist of a photolyase homology region (PHR) carrying the oxidized flavin adenine dinucleotide (FAD) cofactor, and a cryptochrome C-terminal extension (CCT), which is essential for signaling. Absorption of blue/UVA light leads to formation of the FAD neutral radical as the likely signaling state, and ultimately activates the CCT. Little is known about the signal transfer from the flavin to the CCT. Here, we investigated the photoreaction of the PHR by time-resolved step-scan FT-IR spectroscopy complemented by UV–vis spectroscopy. The first spectrum at 500 ns shows major contributions from the FAD anion radical, which is demonstrated to then be protonated by aspartic acid 396 to the neutral radical within 3.5 μs. The analysis revealed the existence of three intermediates characterized by changes in secondary structure. A marked loss of β-sheet structure is observed in the second intermediate evolving with a time constant of 500 μs. This change is accompanied by a conversion of a tyrosine residue, which is identified as the formation of a tyrosine radical in the UV–vis. The only β-sheet in the PHR is located within the α/β subdomain, ∼25 Å away from the flavin. This subdomain has been previously attributed a role as a putative antenna binding site, but is now suggested to have evolved to a component in the signaling of plant cryptochromes by mediating the interaction with the CCT

Microsecond Light-Induced Proton Transfer to Flavin in the Blue Light Sensor Plant Cryptochrome

Plant cryptochromes are blue light photoreceptors that regulate key responses in growth and daily rhythm of plants and might be involved in magnetoreception. They show structural homology to the DNA repair enzyme photolyase and bind flavin adenine dinucleotide as chromophore. Blue light absorption initiates the photoreduction from the oxidized dark state of flavin to the flavin neutral radical, which is the signaling state of the sensor. Previous time-resolved studies of the photoreduction process have been limited to observation of the decay of the radical in the millisecond time domain. We monitored faster, light-induced changes in absorption of an algal cryptochrome covering a spectral range of 375−750 nm with a streak camera setup. Electron transfer from tryptophan to flavin is completed before 100 ns under formation of the flavin anion radical. Proton transfer takes place with a time constant of 1.7 μs leading to the flavin neutral radical. Finally, the flavin radical and a tryptophan neutral radical decay with a time constant >200 μs in the millisecond and second time domain. The microsecond proton transfer has not been observed in animal cryptochromes from insects or photolyases. Furthermore, the strict separation in time of electron and proton transfer is novel in the field of flavin-containing photoreceptors. The reaction rate implies that the proton donor is not in hydrogen bonding distance to the flavin N5. Potential candidates for the proton donor and the involvement of the tryptophan triad are discussed

Blue-Light-Induced Changes in Arabidopsis Cryptochrome 1 Probed by FTIR Difference Spectroscopy^†

Cryptochromes are blue-light photoreceptors that regulate a variety of responses in animals and plants, including circadian entrainment in Drosophila and photomorphogenesis in Arabidopsis. They comprise a photolyase homology region (PHR) of about 500 amino acids and a C-terminal extension of varying length. In the PHR domain, flavin adenine dinucleotide (FAD) is noncovalently bound. The presence of a second chromophore, such as methenyltetrahydrofolate, in animal and plant cryptochromes is still under debate. Arabidopsis cryptochrome 1 (CRY1) has been intensively studied with regard to function and interaction of the protein in vivo and in vitro. However, little is known about the pathway from light absorption to signal transduction on the molecular level. We investigated the full-length CRY1 protein by Fourier transform infrared (FTIR) and UV/vis difference spectroscopy. Starting from the fully oxidized state of the chromophore FAD, a neutral flavoprotein radical is formed upon illumination in the absence of any exogenous electron donor. A preliminary assignment of the chromophore bands is presented. The FTIR difference spectrum reveals only moderate changes in secondary structure of the apoprotein in response to the photoreduction of the chromophore. Deprotonation of an aspartic or glutamic acid, probably D396, accompanies radical formation, as is deduced from the negative band at 1734 cm-1 in D2O. The main positive band at 1524 cm-1 in the FTIR spectrum shows a strong shift to lower frequencies as compared to other flavoproteins. Together with the unusual blue-shift of the absorption in the visible range to 595 nm, this clearly distinguishes the radical form of CRY1 from those of structurally highly homologous DNA photolyases. As a consequence, the direct comparison of cryptochrome to photolyase in terms of photoreactivity and mechanism has to be made with caution

Synthesis of Monodisperse Oligo(1,4-phenyleneethynylene-alt-1,4-triptycyleneethynylene)s

The synthesis of monodisperse oligo(p-phenyleneethynylene)s 8an with alternating 2,5-dihexyl-1,4-phenylene and 6,14-di-tert-butyl-1,4-triptycylene units and orthogonally protected alkyne end groups is reported. Starting from 6,14-di-tert-butyl-1-(2-triisopropylsilylethynyl)-4-(2-trimethylsilylethynyl)triptycene (5a), 1,4-dihexyl-2,5-diiodobenzene (10), and 1,4-dihexyl-2-iodo-5-(3-hydroxyprop-1-ynyl)benzene (9), oligomers with up to four repeating units, i.e., eight phenyleneethynylene units, were prepared through a partially divergent−convergent route with the alkynyl−aryl (Sonogashira−Hagihara) coupling as the key reaction. The starting compound 5a was prepared from triptycenequinone through a sequence of addition of 2-trialkylsilylethynyllithium, reduction and concomitant elimination of water, conversion of the phenol into a triflate, and finally Pd/Cu-catalyzed coupling with trialkylsilylethyne. A similar access to the key compound for a stringent divergent−convergent route, 6,14-di-tert-butyl-1-(3-hydroxybut-1-ynyl)-4-(2-triisopropylsilylethynyl)triptycene (6), is reported. The optical properties of the oligomers 8an and the corresponding oligo(2,5-dihexyl-1,4-phenyleneethynylene)s in dilute solution are almost identical, whereas they differ significantly for the solid, undiluted compounds

Blue-Light-Induced Unfolding of the Jα Helix Allows for the Dimerization of Aureochrome-LOV from the Diatom <i>Phaeodactylum tricornutum</i>

Aureochromes have recently been shown to act as blue-light-regulated transcription factors in the stramenopile alga Vaucheria frigida. They comprise a light-, oxygen-, or voltage-sensitive (LOV) domain as a sensory module with flavin mononucleotide (FMN) as a chromophore and a basic region leucine zipper (bZIP) domain as an effector. Aureochromes are the only members of a large LOV protein family, where the effector domain is located N-terminal to the sensor domain. This domain inversion positions the linking Jα helix of other LOV proteins to the terminus, raising the question of the role of Jα in aureochrome signaling. In phototropins, signaling proceeds from LOV2 via dissociation and unwinding of the Jα helix to the C-terminal kinase effector domain. In contrast, other LOV proteins have been demonstrated to activate the effector without the unfolding of Jα. We investigated the LOV domain of aureochrome1a from the diatom Phaeodactylum tricornutum both with and without the Jα helix. Fourier transform infrared difference spectroscopy provides evidence that the Jα helix unfolds upon illumination. This unfolding is prerequisite for light-induced dimerization of LOV. Under illumination, full conversion to the dimer was observed by size exclusion chromatography. In the absence of the helix, a monomer was detected in the dark and in the light. As a further effect, the recovery of the dark state is 6-fold slower in LOV-Jα than LOV. We therefore postulate that the Jα helix plays an important role in aureochrome signaling

Primary Events in the Blue Light Sensor Plant Cryptochrome: Intraprotein Electron and Proton Transfer Revealed by Femtosecond Spectroscopy

Photoreceptors are chromoproteins that undergo fast conversion from dark to signaling states upon light absorption by the chromophore. The signaling state starts signal transduction in vivo and elicits a biological response. Therefore, photoreceptors are ideally suited for analysis of protein activation by time-resolved spectroscopy. We focus on plant cryptochromes which are blue light sensors regulating the development and daily rhythm of plants. The signaling state of these flavoproteins is the neutral radical of the flavin chromophore. It forms on the microsecond time scale after light absorption by the oxidized state. We apply here femtosecond broad-band transient absorption to early stages of signaling-state formation in a plant cryptochrome from the green alga <i>Chlamydomonas reinhardtii</i>. Transient spectra show (i) subpicosecond decay of flavin-stimulated emission and (ii) further decay of signal until 100 ps delay with <i>nearly</i> constant spectral shape. The first decay (i) monitors electron transfer from a nearby tryptophan to the flavin and occurs with a time constant of τ_ET = 0.4 ps. The second decay (ii) is analyzed by spectral decomposition and occurs with a characteristic time constant τ₁ = 31 ps. We reason that hole transport through a tryptophan triad to the protein surface and partial deprotonation of tryptophan cation radical hide behind τ₁. These processes are probably governed by vibrational cooling. Spectral decomposition is used together with anisotropy to obtain the relative orientation of flavin and the final electron donor. This narrows the number of possible electron donors down to two tryptophans. Structural analysis suggests that a set of histidines surrounding the terminal tryptophan may act as proton acceptor and thereby stabilize the radical pair on a 100 ps time scale

Blue Light Induces Global and Localized Conformational Changes in the Kinase Domain of Full-Length Phototropin

The blue-light photoreceptor phototropin plays a crucial role in optimizing photosynthesis in plants. In the two light-, oxygen-, or voltage-sensitive (LOV) domains of phototropin, the light stimulus is absorbed by the flavin chromophores. The signal is assumed to be transferred via dissociation and unfolding of a conserved Jα helix element to the serine/threonine kinase domain. We investigated full-length phototropin from the green alga Chlamydomonas reinhardtii by Fourier transform infrared spectroscopy to shed light on the signal transfer within the protein and on the structural response of the kinase. Light-induced structural changes were assigned by comparing signals of the full-length protein with those of the truncated LOV1-LOV2-Jα and LOV1-LOV2 and with those of deletion mutants. A loss of helicity originating from the Jα linker helix was observed in LOV1-LOV2-Jα in agreement with previous studies of LOV2-Jα. Full-length phototropin showed reversible global conformational changes via several turn elements. These changes were suppressed in a deletion mutant lacking the Jα linker and are attributed to the kinase domain. The loss of turn structure is interpreted as a light-induced opening of the kinase tertiary structure upon release of the LOV2 domain. Concomitant protonation changes of Asp or Glu residues in the kinase domain were not observed. A light-induced loss in helicity was observed only in the presence of a phototropin-characteristic 54-amino acid extension of the kinase activation loop, which is predicted to be located apart from the catalytic cleft. This response of the extension might play a significant role in the phototropin signaling process

Response of the Sensory Animal-like Cryptochrome aCRY to Blue and Red Light As Revealed by Infrared Difference Spectroscopy

Cryptochromes act as blue light sensors in plants, insects, fungi, and bacteria. Recently, an animal-like cryptochrome (aCRY) was identified in the green alga <i>Chlamydomonas reinhardtii</i> by which gene expression is altered in response to not only blue light but also yellow and red light. This unique response of a flavoprotein <i>in vivo</i> has been attributed to the fact that the neutral radical of the flavin chromophore acts as dark form of the sensor, which absorbs in almost the entire visible spectral range (<680 nm). Here, we investigated light-induced processes in the protein moiety of full-length aCRY by UV–vis and Fourier transform infrared spectroscopy. Findings are compared to published results on the homologous (6-4) photolyases, DNA repair enzymes. The oxidized state of aCRY is converted to the neutral radical by blue light. The recovery is strongly dependent on pH and might be catalyzed by a conserved histidine of the (6-4)/clock cluster. The decay is independent of oxygen concentration in contrast to that of other cryptochromes and (6-4) photolyases. This blue light reaction of the oxidized flavin is not accompanied by any detectable changes in secondary structure, in agreement with a role <i>in vivo</i> of an unphysiological preactivation. In contrast, the conversion by red light of the neutral radical to the anionic fully reduced state proceeds with conformational changes in turn elements, which most probably constitute a part of the signaling process. These changes have not been detected in the corresponding transition of (6-4) photolyase, which points to a decisive difference between the sensor and the enzyme

Primary Events in the Blue Light Sensor Plant Cryptochrome: Intraprotein Electron and Proton Transfer Revealed by Femtosecond Spectroscopy

Photoreceptors are chromoproteins that undergo fast conversion from dark to signaling states upon light absorption by the chromophore. The signaling state starts signal transduction in vivo and elicits a biological response. Therefore, photoreceptors are ideally suited for analysis of protein activation by time-resolved spectroscopy. We focus on plant cryptochromes which are blue light sensors regulating the development and daily rhythm of plants. The signaling state of these flavoproteins is the neutral radical of the flavin chromophore. It forms on the microsecond time scale after light absorption by the oxidized state. We apply here femtosecond broad-band transient absorption to early stages of signaling-state formation in a plant cryptochrome from the green alga Chlamydomonas reinhardtii. Transient spectra show (i) subpicosecond decay of flavin-stimulated emission and (ii) further decay of signal until 100 ps delay with nearly constant spectral shape. The first decay (i) monitors electron transfer from a nearby tryptophan to the flavin and occurs with a time constant of τET = 0.4 ps. The second decay (ii) is analyzed by spectral decomposition and occurs with a characteristic time constant τ1 = 31 ps. We reason that hole transport through a tryptophan triad to the protein surface and partial deprotonation of tryptophan cation radical hide behind τ1. These processes are probably governed by vibrational cooling. Spectral decomposition is used together with anisotropy to obtain the relative orientation of flavin and the final electron donor. This narrows the number of possible electron donors down to two tryptophans. Structural analysis suggests that a set of histidines surrounding the terminal tryptophan may act as proton acceptor and thereby stabilize the radical pair on a 100 ps time scale