Light-induced Conformational Changes Research Articles

The Magnitude of the Light-induced Conformational Change in Different Rhodopsins Correlates with Their Ability to Activate G Proteins

Light converts rhodopsin, the prototypical G protein-coupled receptor, into a form capable of activating G proteins. Recent work has shown that the light-activated state of different rhodopsins can possess different molecular properties, especially different abilities to activate G protein. For example, bovine rhodopsin is approximately 20-fold more effective at activating G protein than parapinopsin, a non-visual rhodopsin, although these rhodopsins share relatively high sequence similarity. Here we have investigated possible structural aspects that might underlie this difference. Using a site-directed fluorescence labeling approach, we attached the fluorescent probe bimane to cysteine residues introduced in the cytoplasmic ends of transmembrane helices V and VI in both rhodopsins. The fluorescence spectra of these probes as well as their accessibility to aqueous quenching agents changed dramatically upon photoactivation in bovine rhodopsin but only moderately so in parapinopsin. We also compared the relative movement of helices V and VI upon photoactivation of both rhodopsins by introducing a bimane label and the bimane-quenching residue tryptophan into helices VI and V, respectively. Both receptors showed movement in this region upon activation, although the movement appears much greater in bovine rhodopsin than in parapinopsin. Together, these data suggest that a larger conformational change in helices V and VI of bovine rhodopsin explains why it has greater G protein activation ability than other rhodopsins. The different amplitude of the helix movement may also be responsible for functional diversity of G protein-coupled receptors.

Blue light photosensors: Examples of environmentally‐regulated protein/protein interactions

Many biological processes are regulated by controlling protein conformation in response to specific environmental cues. This is often mediated by modular protein domains that use environmentally‐triggered changes in bound cofactor conformation or configuration to regulate protein/protein interactions. Photoreceptors exemplify these principles, as they depend on photochemistry mediated by organic chromophores to detect light. In particular, several classes of blue light sensors use flavin chromophores that alter their interactions with the surrounding protein to initiate conformational changes and signaling. We have used solution NMR spectroscopy and other biophysical methods to examine these processes in two classes of photosensory domains.The first of these are Light‐Oxygen‐Voltage (LOV) domains, members of the PAS family of environmental sensors. Illumination generates a covalent protein‐flavin bond within the α/β core of LOV domains, leading to conformational changes in the β‐sheet adjacent to the chromophore. These changes are propagated to non‐canonical protein elements on the other side of this sheet, modifying their function. We will show the generality of this process with data for several classes of LOV‐containing proteins, including serine/threonine kinases, histidine kinases and DNA binding proteins.We will compare these results with parallel studies of BLUF domains (sensors of Blue Light Utilizing FAD), which use different protein topology and photochemistry than LOV domains, but appear to harness light‐induced conformational changes in a similar manner despite these fundamental differences.This work is supported by grants from the NIH (R01 GM081875) and the Robert A. Welch Foundation (I‐1424).

Purification and Characterization of an Activated Rhodopsin/Transducin Complex

Light-induced conformational changes of the dim-light photoreceptor rhodopsin promote efficient binding and activation of the intracellular guanine nucleotide-binding protein, transducin. This activation event initiates GDP/GTP exchange and subsequent dissociation of transducin, from the activated photoreceptor. In this work we have developed a method to assemble and purify an activated rhodopsin/transducin complex in both detergent micelles and lipid bilayers. Activated rhodopsin was immobilized on an affinity resin, allowed to bind to transducin, and extensively washed to remove nonbinding material prior to elution. Evaluation of the eluate by SDS-PAGE and UV-visible absorption spectoscopy confirm the presence of rhodopsin and transducin. The incubation of inactive rhodopsin with transducin in a control experiment resulted in the elution of rhodopsin but no transducin. Activity of the rhodopsin/transducin complex was measured by rhodopsin-dependent GDP release, the uptake of GTPγS, and the nucleotide-dependent release of transducin.

1P-222 青色光センサータンパク質PixDの光誘起構造変化および分子間相互作用変化の研究(光生物-視覚・光受容,第47回日本生物物理学会年会)

1P-222 青色光センサータンパク質PixDの光誘起構造変化および分子間相互作用変化の研究(光生物-視覚・光受容,第47回日本生物物理学会年会)

Light-Induced Dynamics in Photosystem I Electron Transfer

Protein dynamics are likely to play important, regulatory roles in many aspects of photosynthetic electron transfer, but a detailed description of these coupled protein conformational changes has been unavailable. In oxygenic photosynthesis, photosystem I catalyzes the light-driven oxidation of plastocyanin or cytochrome c and the reduction of ferredoxin. A chlorophyll (chl) a/a′ heterodimer, P700, is the secondary electron donor, and the two P700 chl, are designated PA and PB. We used specific chl isotopic labeling and reaction-induced Fourier-transform infrared spectroscopy to assign chl keto vibrational bands to PA and PB. In the cyanobacterium, Synechocystis sp. PCC 6803, the chl keto carbon was labeled from 13C-labeled glutamate, and the chl keto oxygen was labeled from 18O2. These isotope-based assignments provide new information concerning the structure of PA+, which is found to give rise to two chl keto vibrational bands, with frequencies at 1653 and 1687cm−1. In contrast, PA gives rise to one chl keto band at 1638cm−1. The observation of two PA+ keto frequencies is consistent with a protein relaxation-induced distribution in PA+ hydrogen bonding. These results suggest a light-induced conformational change in photosystem I, which may regulate the oxidation of soluble electron donors and other electron-transfer reactions. This study provides unique information concerning the role of protein dynamics in oxygenic photosynthesis.

Application of Fluorescence Resonance Energy Transfer (FRET) to Investigation of Light-Induced Conformational Changes of the Phoborhodopsin/Transducer Complex†

The photoreceptor phoborhodopsin (ppR; also called sensory rhodopsin II) forms a complex with its cognate the Halobacterial transducer II (pHtrII) in the membrane, through which changes in the environmental light conditions are transmitted to the cytoplasm in Natronomonas pharaonis to evoke negative phototaxis. We have applied a fluorescence resonance energy transfer (FRET)-based method for investigation of the light-induced conformational changes of the ppR/pHtrII complex. Several far-red dyes were examined as possible fluorescence donors or acceptors because of the absence of the spectral overlap of these dyes with all the photointermediates of ppR. The flash-induced changes of distances between the donor and an acceptor linked to cysteine residues which were genetically introduced at given positions in pHtrII(1-159) and ppR were determined from FRET efficiency changes. The dye-labeled complex was studied as solubilized in 0.1% n-dodecyl-beta-D-maltoside (DDM). The FRET-derived changes in distances from V78 and A79 in pHtrII to V185 in ppR were consistent with the crystal structure data (Moukhametzianov, R. et al. [2006] Nature, 440, 115-119). The distance from D102 in pHtrII linker region to V185 in ppR increased by 0.33 angstroms upon the flash excitation. These changes arose within 70 ms (the dead time of instrument) and decayed with a rate of 1.1 +/- 0.2 s. Thus, sub-angstrom-scale distance changes in the ppR/pHtrII complex were detected with this FRET-based method using far-red fluorescent dyes; this method should be a valuable tool to investigate conformation changes in the transducer, in particular its dynamics.

NTP-binding properties of the blue-light receptor YtvA and effects of the E105L mutation

YtvA is a blue-light-sensing protein from Bacillus subtilis related to plant phototropins. It carries a LOV (light, oxygen and voltage) domain, binding FMN (flavin mononucleotide) as chromophore, and a STAS (sulphate transporters and antisigma-factor antagonists) domain with poorly characterized function. We have recently shown that YtvA binds triphosphate nucleotides (NTP) and highlighted a structural similarity between the STAS domain and small GTP-binding proteins. In this work we further investigated the NTP-binding properties of YtvA, employing a fluorescent derivative of GTP (GTP(TR)) and mutagenesis experiments. The main results are as follows: (a) competition experiments indicate that the affinity of YtvA for GTP is much higher than that for GDP and GMP. (b) Blue-light-induced structural changes are transmitted from the LOV core to the NTP-binding cavity, establishing a possible intraprotein signal-transduction pathway. (c) A mutation in the central beta-scaffold of the LOV core, E105L, impairs the light-driven spectroscopic changes of bound GTP(TR). This result is supported by circular dichroism data, in that YtvA-E105L does not show the light-induced conformational change in the turn fraction that characterizes YtvA, implying that E105 is functionally important. (d) In the structural model of the LOV-STAS complex, based on docking algorithms, the interface includes the Ibeta-Hbeta loop on the LOV core, as well as parts of the central beta-scaffold. E105 is predicted to interact with the LOV-STAS linker region, suggested to play a role in phototropin signaling.

Dynamics of Light-Induced Conformational Changes of the Phoborhodopsin/Transducer Complex Formed in the n-Dodecyl β-d-Maltoside Micelle

A complex of photoreceptor phoborhodopsin (ppR; also called sensory rhodopsin II) and its cognate halobacterial transducer II (pHtrII) existing in the plasma membrane mediates the light signal to the cytoplasm in the earliest step of negative phototaxis in Natronomonas pharaonis. We have investigated the dynamics of the light-induced conformational changes of the ppR/pHtrII(1-159) complex formed in the presence of 0.1% n-dodecyl beta-d-maltoside (DDM) by a fluorescence resonance energy transfer (FRET) based method. Fluorescence donor and acceptor dyes were linked to cysteine residues genetically introduced at given positions in pHtrII and ppR. The light-induced FRET efficiency changes for various pairs of dye-labeled cysteine residues were determined to examine dynamics of movements of given residues in the transmembrane and the linker region including the HAMP domain in pHtrII induced by photoexcitation of ppR. Upon flash excitation of ppR, FRET efficiency changed depending on pairs of the labeled cysteine residues. The distances between V185 in ppR and the five given residues (102 through 141) in the pHtrII linker region estimated from the FRET efficiency increased by 0.3-0.8 A; on the other hand, the distances between S31 in ppR and the five residues in pHtrII decreased. The changes arose within 70 ms (the dead time of instrument) and decayed at a rate of 1.1 +/- 0.2 s. Azide significantly increased the decay rate of light-induced FRET efficiency changes by accelerating the decay of the M state of ppR. The decay rate of FRET efficiency changes coincided with the rate of recovery of the ppR to the initial state but not the decay of the M state. We conclude that the light-induced conformational change of pHtrII occurs before, at the formation or during the M state, and its relaxation is coupled tightly with the decay of the O state of ppR in the 1:1 complex formed in the DDM micelle.

Analysis of Light-Induced Conformational Changes of Natronomonas pharaonis Sensory Rhodopsin II by Time Resolved Electron Paramagnetic Resonance Spectroscopy†

The nature and kinetics of the conformational changes leading to the activated state of NpSRII/NpHtrII157 were investigated by time-resolved electron paramagnetic resonance (TR-EPR) spectroscopy in combination with site-directed spin labeling (SDSL) on a series of spin labeled mutants of NpSRII. A structural rearrangement of the cytoplasmic moiety of NpSRII upon light activation was detected (helices B, C, F and G). The increase in distance between helices C and F in the M-trapped state of the complex observed in one double mutant is in line with the notion that an outward movement of helix F occurs upon receptor activation. The data obtained from the NpSRII/NpHtrII157 complex reconstituted in purple membrane lipids are compared with those obtained from the X-ray structure of the late M-state of the complex which shows some discrepancies. The results are discussed in the context also of other biophysical and EPR experimental evidences.

2P357 シロイヌナズナphot1におけるLOV2ドメインからJαヘリックスへの光誘起構造変化の伝達メカニズム(光生物学(光合成・視覚と光受容),口頭発表,第45回日本生物物理学会年会)

2P357 シロイヌナズナphot1におけるLOV2ドメインからJαヘリックスへの光誘起構造変化の伝達メカニズム(光生物学(光合成・視覚と光受容),口頭発表,第45回日本生物物理学会年会)

2P360 シロイヌナズナ青色光受容体Phototropin1のLOV2-kinase領域の光誘起構造変化(光生物(視覚・光受容),ポスター発表,第45回日本生物物理学会年会)

2P360 シロイヌナズナ青色光受容体Phototropin1のLOV2-kinase領域の光誘起構造変化(光生物(視覚・光受容),ポスター発表,第45回日本生物物理学会年会)

Conformational analysis of the blue-light sensing protein YtvA reveals a competitive interface for LOV—LOV dimerization and interdomain interactions

The Bacillus subtilis protein YtvA is related to plant phototropins in that it senses UVA-blue-light by means of the flavin binding LOV domain, linked to a nucleotide-binding STAS domain. The structural basis for interdomain interactions and functional regulation are not known. Here we report the conformational analysis of three YtvA constructs, by means of size exclusion chromatography, circular dichroism (CD) and molecular docking simulations. The isolated YtvA-LOV domain (YLOV, aa 25-126) has a strong tendency to dimerize, prevented in full-length YtvA, but still observed in YLOV carrying the N-terminal extension (N-YLOV, aa 1-126). The analysis of CD data shows that both the N-terminal cap and the linker region (aa 127-147) between the LOV and the STAS domain are helical and that the central beta-scaffold is distorted in the LOV domains dimers. The involvement of the central beta-scaffold in dimerization is supported by docking simulation of the YLOV dimer and the importance of this region is highlighted by light-induced conformational changes, emerging from the CD data analysis. In YtvA, the beta-strand fraction is notably less distorted and distinct light-driven changes in the loops/turn fraction are detected. The data uncover a common surface for LOV-LOV and intraprotein interaction, involving the central beta-scaffold, and offer hints to investigate the molecular basis of light-activation and regulation in LOV proteins.

Nanoblossoms: Light-Induced Conformational Changes of Cationic Polyelectrolyte Stars in the Presence of Multivalent Counterions

We analyze the structure of star-shaped polyelectrolytes in the presence of di- and trivalent counterions, and we use the gained knowledge to manipulate the polyelectrolyte's conformation by light. By applying dynamic light scattering and atomic force microscopy, we demonstrate that, at constant ionic strength, the arms of the cationic polyelectrolyte retract when adding multivalent counterions. Adding trivalent hexacyanocobaltate(III) ions leads to a collapse of the polyelectrolyte star even at low concentrations. This is shown by analysis of the star polyelectrolytes in solution as well as in the adsorbed state on mica surfaces. Considerably higher salt concentrations are necessary to obtain the same contraction of the polyelectrolyte star if the divalent tetracyanonickelate(II) ions are used. Sufficiently high multivalent counterion concentration leads finally to the precipitation of the polymer from the solution. We demonstrate that we can switch a polyelectrolyte star from the collapsed to the expanded state by transforming the trivalent hexacyanocobaltate(III) ions into a mixture of mono- and divalent ions by UV light. Thus, these collapsed stars react to light like "nanoblossoms". Moreover, polyelectrolyte stars precipitated through addition of the trivalent hexacyanocobaltate(III) ions can be redissolved by irradiation with light (photoinduced dissolution). Hence, the conformation and interaction of star polyelectrolytes can be switched by light. Possible applications of this novel way of manipulating polymers are discussed.

PAS Domain Allostery and Light-induced Conformational Changes in Photoactive Yellow Protein upon I 2 Intermediate Formation, Probed with Enhanced Hydrogen/Deuterium Exchange Mass Spectrometry

Photoactive yellow protein (PYP) is a small bacterial photoreceptor that undergoes a light-activated reaction cycle. PYP is also the prototypical Per-Arnt-Sim (PAS) domain. PAS domains, found in diverse multi-domain proteins from bacteria to humans, mediate protein–protein interactions and function as sensors and signal transducers. Here, we investigate conformational and dynamic changes in solution in wild-type PYP upon formation of the long-lived putative signaling intermediate I 2 with enhanced hydrogen/deuterium exchange mass spectrometry (DXMS). The DXMS results showed that the central β-sheet remains stable but specific external protein segments become strongly deprotected. Light-induced disruption of the dark-state hydrogen bonding network in I 2 produces increased flexibility and opening of PAS core helices α3 and α4, releases the β4-β5 hairpin, and propagates conformational changes to the central β-sheet. Surprisingly, the first ∼10 N-terminal residues, which are essential for fast dark-state recovery from I 2, become more protected. By combining the DXMS results with our crystallographic structures, which reveal detailed changes near the chromophore but limited protein conformational change, we propose a mechanism for I 2 state formation. This mechanism integrates the results from diverse biophysical studies of PYP, and links an allosteric T to R-state conformational transition to three pathways for signal propagation within the PYP fold. On the basis of the observed changes in PYP plus commonalities shared among PAS domain proteins, we further propose that PAS domains share this conformational mechanism, which explains the versatile signal transduction properties of the structurally conserved PYP/PAS module by framework-encoded allostery.

Uncoupling of Electron and Proton Transfers in the Photocycle of Bacterial Reaction Centers under High Light Intensity

Photosynthetic reaction centers produce and export oxidizing and reducing equivalents in expense of absorbed light energy. The formation of fully reduced quinone (quinol) requires a strict (1:1) stoichiometric ratio between the electrons and H(+) ions entering the protein. The steady-state rates of both transports were measured separately under continuous illumination in the reaction center from the photosynthetic bacterium Rhodobacter sphaeroides. The uptake of the first proton was retarded by different methods and made the rate-limiting reaction in the photocycle. As expected, the rate constant of the observed proton binding remained constant (7 s(-)(1)), but that of the cytochrome photooxidation did show a remarkably large increase from 14 to 136 s(-)(1) upon increase of the exciting light intensity up to 5 W/cm(2) (808 nm) at pH 8.4 in the presence of NiCl(2). This corresponds to about 20:1 (e(-):H(+)) stoichiometric ratio. The observed enhancement is linearly proportional to the light intensity and the rate constant of the proton uptake by the acceptor complex and shows saturation character with quinone availability. For interpretation of the acceleration of cytochrome turnover, an extended model of the photocycle is proposed. A fraction of photochemically trapped RC can undergo fast (>10(3) s(-)(1)) conformational change where the semiquinone loses its high binding affinity (the dissociation constant increases by more than 5 orders of magnitude) and dissociates from the Q(B) binding site of the protein with a high rate of 4000 s(-)(1). Concomitantly, superoxide is being produced. No H(+) ion is taken up, and no quinol is created by the photocycle which is operating in about 25% of the reaction centers at the highest light intensity (5500 s(-)(1)) and slowest proton uptake (3.5 s(-)(1)) used in our experiments. The possible physical background of the light-induced conformational change and the relationship between the energies of dissociation and redox changes of the quinone in the Q(B) binding sites are discussed.

Residues crucial for maintaining short paths in network communication mediate signaling in proteins

Here, we represent protein structures as residue interacting networks, which are assumed to involve a permanent flow of information between amino acids. By removal of nodes from the protein network, we identify fold centrally conserved residues, which are crucial for sustaining the shortest pathways and thus play key roles in long-range interactions. Analysis of seven protein families (myoglobins, G-protein-coupled receptors, the trypsin class of serine proteases, hemoglobins, oligosaccharide phosphorylases, nuclear receptor ligand-binding domains and retroviral proteases) confirms that experimentally many of these residues are important for allosteric communication. The agreement between the centrally conserved residues, which are key in preserving short path lengths, and residues experimentally suggested to mediate signaling further illustrates that topology plays an important role in network communication. Protein folds have evolved under constraints imposed by function. To maintain function, protein structures need to be robust to mutational events. On the other hand, robustness is accompanied by an extreme sensitivity at some crucial sites. Thus, here we propose that centrally conserved residues, whose removal increases the characteristic path length in protein networks, may relate to the system fragility.

2P328 Effect of azide on dynamics of light-induced conformational change of phoborhodopsin/transducer complex(42. Sensory signal transduction,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

2P328 Effect of azide on dynamics of light-induced conformational change of phoborhodopsin/transducer complex(42. Sensory signal transduction,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Low-Temperature Interquinone Electron Transfer in Photosynthetic Reaction Centers fromRhodobacter sphaeroidesandBlastochloris viridis: Characterization of QB-States by High-Frequency Electron Paramagnetic Resonance (EPR) and Electron−Nuclear Double Resonance (ENDOR)

High-frequency electron paramagnetic resonance (HF EPR) techniques have been employed to look for localized light-induced conformational changes in the protein environments around the reduced secondary quinone acceptor (Q(B)(-)) in Rhodobacter sphaeroides and Blastochloris viridis RCs. The Q(A)(-) and Q(B)(-) radical species in Fe-removed/Zn-replaced protonated RCs substituted with deuterated quinones are distinguishable with pulsed D-band (130 GHz) EPR and provide native probes of both the low-temperature Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron-transfer event and the structure of trapped conformational substates. We report here the first spectroscopic evidence that cryogenically trapped, light-induced changes enable low-temperature Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer in the B. viridis RC and the first observation of an inactive, trapped P(+)Q(B)(-) state in both R. sphaeroides and B. viridis RCs that does not recombine at 20 K. The high resolution and orientational selectivity of HF electron-nuclear double resonance (ENDOR) allows us to directly probe protein environments around Q(B)(-) for distinct P(+)Q(B)(-) kinetic RC states by spectrally selecting specific nuclei in isotopically labeled samples. No structural differences in the protein structure near Q(B)(-) or reorientation (within 5 degrees ) of Q(B)(-) was observed with HF ENDOR spectra of two states of P(+)Q(B)(-): "active" and "inactive" states with regards to low-temperature electron transfer. These results reveal a remarkably enforced local protein environment for Q(B) in its reduced semiquinone state and suggest that the conformational change that controls reactivity resides beyond the Q(B) local environment.

Sterically Locked Synthetic Bilin Derivatives and Phytochrome Agp1 from Agrobacterium tumefaciens Form Photoinsensitive Pr- and Pfr-like Adducts

Phytochrome photoreceptors undergo reversible photoconversion between the red-absorbing form, Pr, and the far-red-absorbing form, Pfr. The first step in the conversion from Pr to Pfr is a Z to E isomerization around the C15=C16 double bond of the bilin chromophore. We prepared four synthetic biliverdin (BV) derivatives in which rings C and D are sterically locked by cyclizing with an additional carbon chain. In these chromophores, which are termed 15Za, 15Zs, 15Ea, and 15Es, the C15=C16 double bond is in either the Z or E configuration and the C14-C15 single bond in either the syn or anti conformation. The chromophores were assembled with Agrobacterium phytochrome Agp1, which incorporates BV as natural chromophore. All locked BV derivatives bound covalently to the protein and formed adducts with characteristic spectral properties. The 15Za adduct was spectrally similar to the Pr form and the 15Ea adduct similar to the Pfr form of the BV adduct. Thus, the chromophore of Agp1 adopts a C15=C16 Z configuration and a C14-C15 anti conformation in the Pr form and a C15=C16 E configuration and a C14-C15 anti conformation in the Pfr form. Both the 15Zs and the 15Es adducts absorbed only in the blue region of the visible spectra. All chromophore adducts were analyzed by size exclusion chromatography and histidine kinase activity to probe for protein conformation. In either case, the 15Za adduct behaved like the Pr and the 15Ea adduct like the Pfr form of Agp1. Replacing the natural chromophore by a locked 15Ea derivative can thus bring phytochrome holoprotein in the Pfr form in darkness. In this way, physiological action of Pfr can be studied in vivo and separated from Pr/Pfr cycling and other light effects.

Biochemical and Functional Characterization of BLUF-Type Flavin-Binding Proteins of Two Species of Cyanobacteria

BLUF (a sensor of Blue-Light Using FAD) is a novel putative photoreceptor domain that is found in many bacteria and some eukaryotic algae. As found on genome analysis, certain cyanobacteria have BLUF proteins with a short C-terminal extension. As typical examples, Tll0078 from thermophilic Thermosynechococcus elongatus BP-1 and Slr1694 from mesophilic Synechocystis sp. PCC 6803 were comparatively studied. FAD of both proteins was hardly reduced by exogenous reductants or mediators except methylviologen but showed a typical spectral shift to a longer wavelength upon excitation with blue light. In particular, freshly prepared Tll0078 protein showed slow but reversible aggregation, indicative of light-induced conformational changes in the protein structure. Tll0078 is far more stable as to heat treatment than Slr1694, as judged from flavin fluorescence. The slr1694-disruptant showed phototactic motility away from the light source (negative phototaxis), while the wild type Synechocystis showed positive phototaxis toward the source. Yeast two-hybrid screening with slr1694 showed self-interaction of Slr1694 (PixD) with itself and interaction with a novel PatA-like response regulator, Slr1693 (PixE). These results were discussed in relation to the signaling mechanism of the "short" BLUF proteins in the regulation of cyanobacterial phototaxis.