Light-induced Conformational Changes Research Articles

Mechanism of Photosignaling by Drosophila Cryptochrome: ROLE OF THE REDOX STATUS OF THE FLAVIN CHROMOPHORE

Cryptochrome (CRY) is the primary circadian photoreceptor in Drosophila. Upon light absorption, dCRY undergoes a conformational change that enables it to bind to Timeless (dTIM), as well as to two different E3 ligases that ubiquitylate dTIM and dCRY, respectively, resulting in their proteolysis and resetting the phase of the circadian rhythm. Purified dCRY contains oxidized flavin (FADox), which is readily photoreduced to the anionic semiquinone through a set of 3 highly conserved Trp residues (Trp triad). The crystal structure of dCRY has revealed a fourth Trp (Trp-536) as a potential electron donor. Previously, we reported that the Trp triad played no role in photoinduced proteolysis of dCRY in Drosophila cells. Here we investigated the role of the Trp triad and Trp-536, and the redox status of the flavin on light-induced proteolysis of both dCRY and dTIM and resetting of the clock. We found that both oxidized (FADox) and reduced (FAD) forms of dCRY undergo light-induced conformational change in vitro that enable dCRY to bind JET and that Trp triad and Trp-536 mutations that block known or presumed intraprotein electron transfer reactions do not affect dCRY phototransduction under bright or dim light in vivo as measured by light-induced proteolysis of dCRY and dTIM in Drosophila S2R+ cells. We conclude that both oxidized and reduced forms of dCRY are capable of photosignaling.

Mapping of the local environmental changes in proteins by cysteine scanning.

Protein conformational changes, which regulate the activity of proteins, are induced by the alternation of intramolecular interactions. Therefore, the detection of the local environmental changes around the key amino acid residues is essential to understand the activation mechanisms of functional proteins. Here we developed the methods to scan the local environmental changes using the vibrational band of cysteine S-H group. We validated the sensitivity of this method using bathorhodopsin, a photoproduct of rhodopsin trapped at liquid nitrogen temperature, which undergoes little conformational changes from the dark state as shown by the X-ray crystallography. The cysteine residues were individually introduced into 15 positions of Helix III, which contains several key amino acid residues for the light-induced conformational changes of rhodopsin. The shifts of S-H stretching modes of these cysteine residues and native cysteine residues upon the formation of bathorhodopsin were measured by Fourier transform infrared spectroscopy. While most of cysteine residues demonstrated no shift of S-H stretching mode, cysteine residues introduced at positions 117, 118, and 122, which are in the vicinity of the chromophore, demonstrated the significant changes. The current results are consistent with the crystal structure of bathorhodopsin, implying that the cysteine scanning is sensitive enough to detect the tiny conformational changes.

Light-induced Conformational Changes of LOV1 (Light Oxygen Voltage-sensing Domain 1) and LOV2 Relative to the Kinase Domain and Regulation of Kinase Activity in Chlamydomonas Phototropin

Phototropin (phot), a blue light (BL) receptor in plants, has two photoreceptive domains named LOV1 and LOV2 as well as a Ser/Thr kinase domain (KD) and acts as a BL-regulated protein kinase. A LOV domain harbors a flavin mononucleotide that undergoes a cyclic photoreaction upon BL excitation via a signaling state in which the inhibition of the kinase activity by LOV2 is negated. To understand the molecular mechanism underlying the BL-dependent activation of the kinase, the photochemistry, kinase activity, and molecular structure were studied with the phot of Chlamydomonas reinhardtii. Full-length and LOV2-KD samples of C. reinhardtii phot showed cyclic photoreaction characteristics with the activation of LOV- and BL-dependent kinase. Truncation of LOV1 decreased the photosensitivity of the kinase activation, which was well explained by the fact that the signaling state lasted for a shorter period of time compared with that of the phot. Small angle x-ray scattering revealed monomeric forms of the proteins in solution and detected BL-dependent conformational changes, suggesting an extension of the global molecular shapes of both samples. Constructed molecular model of full-length phot based on the small angle x-ray scattering data proved the arrangement of LOV1, LOV2, and KD for the first time that showed a tandem arrangement both in the dark and under BL irradiation. The models suggest that LOV1 alters its position relative to LOV2-KD under BL irradiation. This finding demonstrates that LOV1 may interact with LOV2 and modify the photosensitivity of the kinase activation through alteration of the duration of the signaling state in LOV2.

Dynamic Solvation and Coupling of the Hydration Shell of ZnII-Substituted Cytochrome c in the Presence of Guanidinium Ions

The fluorescence Stokes shift (FSS) response of Zn(II)-substituted cytochrome c (ZnCytc) is transformed from a monotonic red-shifting response in water to a bidirectional response with much slower time constants in the presence of low concentrations of guanidinium (Gdm(+)) ions. The FSS response in water observed over the 100 ps to 10 ns range has two exponential components with time constants of 135 ps and 1.6 ns that account for a total shift of 30 cm(-1), about one-half of the solvation reorganization energy. In contrast, in the presence of only 0.25 M Gdm(+), the FSS response initially shifts 21 cm(-1) to the blue with a 820 ps time constant and then shifts 60 cm(-1) back to the red with a 3.5 ns time constant. The effect of Gdm(+) on the FSS response effectively saturates at 1.0 M, well below the 1.75 M midpoint of the two-state unfolding transition. These results establish that the FSS response in ZnCytc includes a significant contribution from the surrounding hydration shell, which assumes a perturbed hydrogen-bonding network owing to the binding of Gdm(+) ions to the protein surface. The blue-shifting part of the FSS response arises from a light-induced conformational change that expands the protein- and solvent-derived cavity around the excited-state Zn(II) porphyrin. This non-polar part of the solvation response is enhanced in the presence of Gdm(+) because the protein/solvent surroundings of the Zn(II) porphyrin are effectively more flexible than in water. The enhanced flexibility in the presence of Gdm(+) increases the amplitudes and accordingly lengthens the correlation time scales for the protein and hydration-shell fluctuations that contribute to the FSS response.

Azobenzamide-based proteomorphous objects as a light/pH-induced photoswitchable module

Azobenzamide-based molecular scaffolds viz. (E)-4,4′(diazene-1,2-diyl)bis(N-pyridin-3-yl)benzamide (1) and (E)-4,4′(diazene-1,2-diyl)bis(N-pyridin-4-yl)benzamide (2), yield proteomorphous architectures i.e., secondary “β-pleated sheet” and tertiary “β-hairpin” respectively, that offer “optical data storage” competence as a result of reversible light-induced conformational changes in organic solvent.

Chlorophyll a fluorescence: beyond the limits of the QA model

Chlorophyll a fluorescence is a non-invasive tool widely used in photosynthesis research. According to the dominant interpretation, based on the model proposed by Duysens and Sweers (1963, Special Issue of Plant and Cell Physiology, pp 353-372), the fluorescence changes reflect primarily changes in the redox state of Q(A), the primary quinone electron acceptor of photosystem II (PSII). While it is clearly successful in monitoring the photochemical activity of PSII, a number of important observations cannot be explained within the framework of this simple model. Alternative interpretations have been proposed but were not supported satisfactorily by experimental data. In this review we concentrate on the processes determining the fluorescence rise on a dark-to-light transition and critically analyze the experimental data and the existing models. Recent experiments have provided additional evidence for the involvement of a second process influencing the fluorescence rise once Q(A) is reduced. These observations are best explained by a light-induced conformational change, the focal point of our review. We also want to emphasize that-based on the presently available experimental findings-conclusions on α/ß-centers, PSII connectivity, and the assignment of FV/FM to the maximum PSII quantum yield may require critical re-evaluations. At the same time, it has to be emphasized that for a deeper understanding of the underlying physical mechanism(s) systematic studies on light-induced changes in the structure and reaction kinetics of the PSII reaction center are required.

Light-Induced Subunit Dissociation by a Light–Oxygen–Voltage Domain Photoreceptor from Rhodobacter sphaeroides

Light-oxygen-voltage (LOV) domains bind a flavin chromophore to serve as blue light sensors in a wide range of eukaryotic and prokaryotic proteins. LOV domains are associated with a variable effector domain or a separate protein signaling partner to execute a wide variety of functions that include regulation of kinases, generation of anti-sigma factor antagonists, and regulation of circadian clocks. Here we present the crystal structure, photocycle kinetics, association properties, and spectroscopic features of a full-length LOV domain protein from Rhodobacter sphaeroides (RsLOV). RsLOV exhibits N- and C-terminal helical extensions that form an unusual helical bundle at its dimer interface with some resemblance to the helical transducer of sensory rhodopsin II. The blue light-induced conformational changes of RsLOV revealed from a comparison of light- and dark-state crystal structures support a shared signaling mechanism of LOV domain proteins that originates with the light-induced formation of a flavin-cysteinyl photoadduct. Adduct formation disrupts hydrogen bonding in the active site and propagates structural changes through the LOV domain core to the N- and C-terminal extensions. Single-residue variants in the active site and dimer interface of RsLOV alter photoadduct lifetimes and induce structural changes that perturb the oligomeric state. Size exclusion chromatography, multiangle light scattering, small-angle X-ray scattering, and cross-linking studies indicate that RsLOV dimerizes in the dark but, upon light excitation, dissociates into monomers. This light-induced switch in oligomeric state may prove to be useful for engineering molecular associations in controlled cellular settings.

Blue Light-Induced Conformational Changes in a Light-Regulated Transcription Factor, Aureochrome-1

Aureochrome-1 (AUREO1) is a blue light (BL) receptor that mediates the branching response in the stramenopile alga, Vaucheria frigida. AUREO1 harbors a basic leucine zipper (bZIP) domain at the N-terminus and a light-oxygen-voltage-sensing (LOV) domain within the C-terminal region, and has been suggested to function as a light-regulated transcription factor. To understand the molecular mechanism of AUREO1, we have prepared three recombinant proteins: a full-length AUREO1 (FL), an N-terminal truncated construct containing bZIP and LOV (ZL) and a LOV-only (LOV) construct. The constructs showed the same absorption and fluorescent spectra in the dark state and underwent the characteristic cyclic reaction as previously observed in LOV domains upon BL excitation. FL and ZL bound to DNA in a sequence-specific manner. BL appeared to induce a shift of the α-helical structure of the LOV domain to a β-sheet structure, but did not alter the hydrodynamic radius (R(H)) of this domain. ZL formed a dimer possibly through disulfide linkages in the bZIP and the linker region between bZIP and LOV. BL induced an approximately 5% increase in the R(H) of ZL, although its secondary structure was unchanged. These results support a schema where BL-induced changes in the LOV domain may cause conformational changes in the bZIP and/or the linker of a dimeric ZL molecule. Since a 5% increase of the R(H) was also observed with the FL construct, BL may induce global conformational changes similar to those observed for ZL, and formation of the FL dimer may facilitate DNA binding.

Effects of dehydration on light-induced conformational changes in bacterial photosynthetic reaction centers probed by optical and differential FTIR spectroscopy

Following light-induced electron transfer between the primary donor (P) and quinone acceptor (QA) the bacterial photosynthetic reaction center (RC) undergoes conformational relaxations which stabilize the primary charge separated state P+QA−. Dehydration of RCs from Rhodobacter sphaeroides hinders these conformational dynamics, leading to acceleration of P+QA− recombination kinetics [Malferrari et al., J. Phys. Chem. B 115 (2011) 14732-14750]. To clarify the structural basis of the conformational relaxations and the involvement of bound water molecules, we analyzed light-induced P+QA−/PQA difference FTIR spectra of RC films at two hydration levels (relative humidity r=76% and r=11%). Dehydration reduced the amplitude of bands in the 3700–3550cm−1 region, attributed to water molecules hydrogen bonded to the RC, previously proposed to stabilize the charge separation by dielectric screening [Iwata et al., Biochemistry 48 (2009) 1220–1229]. Other features of the FTIR difference spectrum were affected by partial depletion of the hydration shell (r=11%), including contributions from modes of P (9-keto groups), and from NH or OH stretching modes of amino acidic residues, absorbing in the 3550–3150cm−1 range, a region so far not examined in detail for bacterial RCs. To probe in parallel the effects of dehydration on the RC conformational relaxations, we analyzed by optical absorption spectroscopy the kinetics of P+QA− recombination following the same photoexcitation used in FTIR measurements (20s continuous illumination). The results suggest a correlation between the observed FTIR spectral changes and the conformational rearrangements which, in the hydrated system, strongly stabilize the P+QA− charge separated state over the second time scale.

Time-resolved crystallography for protein structure: the case of heme proteins

Time-resolved Laue diffraction is an elegant approach by which polychromatic X-ray pulses are used in combination with rapid laser triggering of light-sensitive samples so as to make a movie of protein reaction dynamics in real time. The current state-of-the-art with protein Laue diffraction has been time-resolved studies of light-induced conformational changes in myoglobin-CO with 150 ps resolution. Myoglobin has traversed the whole history of pump–probe Laue diffraction, yielding a massive amount of data that have provided considerable insight into the understanding of protein dynamics. A picture of factors governing myoglobin dynamics following ligand dissociation has been drawn from data arising from wild-type and mutant proteins. Other heme proteins have been studied using this approach: Scapharca inequivalvis dimeric hemoglobin, the FixL PAS domain and the photosynthetic reaction center complex of Blastochloris viridis.

Acute depletion of plasma membrane phospholipids—dissecting the roles of PtdIns(4)P and PtdIns(4,5)P2

Acute depletion of plasma membrane phospholipids—dissecting the roles of PtdIns(4)P and PtdIns(4,5)P2

A PixD—PapB Chimeric Protein Reveals the Function of the BLUF Domain C-Terminal α-Helices for Light Signal Transduction

Blue light-using flavin (BLUF) proteins form a subfamily of blue light photoreceptors, are found in many bacteria and algae, and are further classified according to their structures. For one type of BLUF-containing protein, e.g. PixD, the central axes of its two C-terminal α-helices are perpendicular to the β-sheet of its N-terminal BLUF domain. For another type, e.g. PapB, the central axes of its two C-terminal α-helices are parallel to its BLUF domain β-sheet. However, the functional significance of the different orientations with respect to phototransduction is not clear. For the study reported herein, we constructed a chimeric protein, Pix0522, containing the core of the PixD BLUF domain and the C-terminal region of PapB, including the two α-helices, and characterized its biochemical and spectroscopic properties. Fourier transform infrared spectroscopy detected similar light-induced conformational changes in the C-terminal α-helices of Pix0522 and PapB. Pix0522 interacts with and activates the PapB-interacting enzyme, PapA, demonstrating the functionality of Pix0522. These results provide direct evidence that the BLUF C-terminal α-helices function as an intermediary that accepts the flavin-sensed blue light signal and transmits it downstream during phototransduction.

The β-Scaffold of the LOV Domain of the Brucella Light-Activated Histidine Kinase Is a Key Element for Signal Transduction

Light–oxygen–voltage (LOV) domains are blue-light-activated signaling modules present in a wide range of sensory proteins. Among them, the histidine kinases are the largest group in prokaryotes (LOV-HK). Light modulates the virulence of the pathogenic bacteria Brucella abortus through LOV-HK. One of the striking characteristic of Brucella LOV-HK is the fact that the protein remains activated upon light sensing, without recovering the basal state in the darkness. In contrast, the light state of the isolated LOV domain slowly returns to the dark state. To gain insight into the light activation mechanism, we have characterized by X-ray crystallography and solution NMR spectroscopy the structure of the LOV domain of LOV-HK in the dark state and explored its light-induced conformational changes. The LOV domain adopts the α/β PAS (PER-ARNT-SIM) domain fold and binds the FMN cofactor within a conserved pocket. The domain dimerizes through the hydrophobic β-scaffold in an antiparallel way. Our results point to the β-scaffold as a key element in the light activation, validating a conserved structural basis for light-to-signal propagation in LOV proteins.

Crystal Structures of Aureochrome1 LOV Suggest New Design Strategies for Optogenetics

Aureochrome1, a signaling photoreceptor from a eukaryotic photosynthetic stramenopile, confers blue-light-regulated DNA binding on the organism. Its topology, in which a C-terminal LOV sensor domain is linked to an N-terminal DNA-binding bZIP effector domain, contrasts with the reverse sensor-effector topology in most other known LOV-photoreceptors. How, then, is signal transmitted in Aureochrome1? The dark- and light-state crystal structures of Aureochrome1 LOV domain (AuLOV) show that its helical N- and C-terminal flanking regions are packed against the external surface of the core β sheet, opposite to the FMN chromophore on the internal surface. Light-induced conformational changes occur in the quaternary structure of the AuLOV dimer and in Phe298 of the Hβ strand in the core. The properties of AuLOV extend the applicability of LOV domains as versatile design modules that permit fusion to effector domains via either the N- or C-termini to confer blue-light sensitivity.

3B0936 PYPの光構造変化の機械的な制御(蛋白質-構造機能相関III:動態,生体リズム,口頭発表,日本生物物理学会第50回年会(2012年度))

3B0936 PYPの光構造変化の機械的な制御(蛋白質-構造機能相関III:動態,生体リズム,口頭発表,日本生物物理学会第50回年会(2012年度))

A Transporter Converted into a Sensor, a Phototaxis Signaling Mutant of Bacteriorhodopsin at 3.0 Å

Bacteriorhodopsin (BR) and sensory rhodopsin II (SRII), homologous photoactive proteins in haloarchaea, have different molecular functions. BR is a light-driven proton pump, whereas SRII is a phototaxis receptor that transmits a light-induced conformational change to its transducer HtrII. Despite these distinctly different functions, a single residue substitution, Ala215 to Thr215 in the BR retinal-binding pocket, enables its photochemical reactions to transmit signals to HtrII and mediate phototaxis. We pursued a crystal structure of the signaling BR mutant (BR_A215T) to determine the structural changes caused by the A215T mutation and to assess what new photochemistry is likely to be introduced into the BR photoactive site. We crystallized BR_A215T from bicelles and solved its structure to 3.0 Å resolution to enable an atomic-level comparison. The analysis was complemented by molecular dynamics simulation of BR mutated in silico. Three main conclusions regarding the roles of photoactive site residues in signaling emerge from the comparison of BR_A215T, BR, and SRII structures: (i) the Thr215 residue in signaling BR is positioned nearly identically with respect to the retinal chromophore as in SRII, consistent with its role in producing a steric conflict with the retinal C14 group during photoisomerization, proposed earlier to be essential for SRII signaling from vibrational spectroscopy and motility measurements; (ii) Tyr174–Thr204 hydrogen bonding, critical in SRII signaling and mimicked in signaling BR, is likely auxiliary, for example, to maintain Thr204 in the proper position for the steric trigger to occur; and (iii) the primary role of Arg72 in SRII is spectral tuning and not signaling.

Illuminating the early signaling pathway of a fungal light‐oxygen‐voltage photoreceptor

Circadian clocks are molecular timekeepers encountered in a wide variety of organisms, which allow to adapt the cell's metabolism and behavior to the daily and seasonal periods. Their function is regulated by light-sensing proteins, among which Vivid, a light-oxygen-voltage (LOV) sensitive domain of the fungus Neurospora crassa, constitutes one of the most prominent examples. Although the major photochemical and structural changes during the photocycle of this photosensor have been elucidated through experimental means, its signal transduction pathway is still poorly resolved at the molecular level. In this article, we show through molecular dynamics simulation that the primary steps after adduct formation involve a switch of Gln182 in vicinity of the chromophore FAD (flavin-adenine-dinucleotide), followed by a coupling between the Iβ- and Hβ-strands through H-bond formation between Gln182 and Asn161 as well as subsequent weakening of the H-bonding interaction between the Iβ- and Aβ-strands. These processes then induce a reorientation of the Aβ-Bβ-loop with respect to the protein core as well as a simultaneous contraction of the partially unfolded α-helix onto the α-Aβ-linker at the Ncap. Finally, we demonstrate through additional dimer simulations that the light-induced conformational changes, observed in the monomeric case, play a decisive role in controlling the dimerization tendency of Vivid with its partner domains and that the light-state homodimer shows a much larger affinity for aggregation than the dark state.

Temperature-scan cryocrystallography reveals reaction intermediates in bacteriophytochrome

Light is a fundamental signal that regulates important physiological processes such as development and circadian rhythm in living organisms. Phytochromes form a major family of photoreceptors responsible for red light perception in plants, fungi and bacteria. They undergo reversible photoconversion between red-absorbing (Pr) and far-red-absorbing (Pfr) states, thereby ultimately converting a light signal into a distinct biological signal that mediates subsequent cellular responses. Several structures of microbial phytochromes have been determined in their dark-adapted Pr or Pfr states. However, the structural nature of initial photochemical events has not been characterized by crystallography. Here we report the crystal structures of three intermediates in the photoreaction of Pseudomonas aeruginosa bacteriophytochrome (PaBphP). We used cryotrapping crystallography to capture intermediates, and followed structural changes by scanning the temperature at which the photoreaction proceeded. Light-induced conformational changes in PaBphP originate in ring D of the biliverdin (BV) chromophore, and E-to-Z isomerization about the C(15) = C(16) double bond between rings C and D is the initial photochemical event. As the chromophore relaxes, the twist of the C(15) methine bridge about its two dihedral angles is reversed. Structural changes extend further to rings B and A, and to the surrounding protein regions. These data indicate that absorption of a photon by the Pfr state of PaBphP converts a light signal into a structural signal via twisting and untwisting of the methine bridges in the linear tetrapyrrole within the confined protein cavity.

Light-Induced Conformational Changes in Full-Length Arabidopsis thaliana Cryptochrome

Cryptochromes (CRYs) are widespread flavoproteins with homology to photolyases (PHRs), a class of blue-light-activated DNA repair enzymes. Unlike PHRs, both plant and animal CRYs have a C-terminal domain. This cryptochrome C-terminal (CCT) domain mediates interactions with other proteins, while the PHR-like domain converts light energy into a signal via reduction and radical formation of the flavin adenine dinucleotide cofactor. However, the mechanism by which the PHR-like domain regulates the CCT domain is not known. Here, we applied the pulsed-laser-induced transient grating method to detect conformational changes induced by blue-light excitation of full-length Arabidopsis thaliana cryptochrome 1 ( AtCRY1). A significant reduction in the diffusion coefficient of AtCRY1 was observed upon photoexcitation, indicating that a large conformational change occurs in this monomeric protein. AtCRY1 containing a single mutation (W324F) that abolishes an intra-protein electron transfer cascade did not exhibit this conformational change. Moreover, the conformational change was much reduced in protein lacking the CCT domain. Thus, we conclude that the observed large conformational changes triggered by light excitation of the PHR-like domain result from C-terminal domain rearrangement. This inter-domain modulation would be critical for CRYs' ability to transduce a blue-light signal into altered protein–protein interactions for biological activity. Lastly, we demonstrate that the transient grating technique provides a powerful method for the direct observation and understanding of photoreceptor dynamics.

Structure of a Light-Activated LOV Protein Dimer That Regulates Transcription

Light, oxygen, or voltage (LOV) protein domains are present in many signaling proteins in bacteria, archaea, protists, plants, and fungi. The LOV protein VIVID (VVD) of the filamentous fungus Neurospora crassa enables the organism to adapt to constant or increasing amounts of light and facilitates proper entrainment of circadian rhythms. Here, we determined the crystal structure of the fully light-adapted VVD dimer and reveal the mechanism by which light-driven conformational change alters the oligomeric state of the protein. Light-induced formation of a cysteinyl-flavin adduct generated a new hydrogen bond network that released the amino (N) terminus from the protein core and restructured an acceptor pocket for binding of the N terminus on the opposite subunit of the dimer. Substitution of residues critical for the switch between the monomeric and the dimeric states of the protein had profound effects on light adaptation in Neurospora. The mechanism of dimerization of VVD provides molecular details that explain how members of a large family of photoreceptors convert light responses to alterations in protein-protein interactions.