ABSORPTION AND PHOTOCHEMISTRY OF SENSORY RHODOPSIN—I: pH EFFECTS

Pyranine (8-hydroxyl-1,3,6-pyrene-trisulfonate) was used as a pH-probe to test whether there is a light-induced proton release to the bulk phase during the photochemical reaction cycle of sensory rhodopsin-I (SR-I). We conclude that the retinylidene Schiff-base proton is retained by SR-I-containing envelope vesicles during the SR-I photocycle under the conditions described here. Bacteriorhodopsin containing vesicles were used as a control to show that light-induced proton release can be observed under identical data acquisition parameters as those used for SR-I-containing vesicles. In addition, the effects of extravesicular pH on the absorption maximum (lambda max) and the SR-I photocycle were studied. SR-I properties are insensitive to pH in the range approximately 3 to approximately 8 with lambda max remaining at 587 nm. The lambda max shifts to 565 nm below pH 3.0 and to 552 nm at pH 10.8 with an apparent pKa of 8.5. Flash-induced absorbance changes of SR-I are described under neutral, alkaline and acidic conditions. The neutral, alkaline and acid SR-I forms each undergo similar photoreactions producing long-lived (> 500 ms decay half-time) blue-shifted intermediates. The UV/near-UV absorption of the photoproducts from neutral and alkaline SR-I indicate a deprotonated Schiff base, whereas acid SR-I produces a species with lambda max > 460 nm indicative of a protonated Schiff base.     

TRANSFORMATION OF A BOP-HOP-SOP-I-SOP-II-Halobacterium halobium MUTANT TO BOP+: EFFECTS OF BACTERIORHODOPSIN PHOTOACTIVATION ON CELLULAR PROTON FLUXES AND SWIMMING BEHAVIOR

We have transformed Pho81, a Halobacterium halobium mutant strain which does not contain any of the four retinylidene proteins known in this species, with the bop gene cluster to create Pho81BR, a BR+HR-SR-I-SR-II-strain. The absorption spectrum, pigment reconstitution process, light-dark adaptation and photochemical reaction cycle of the expressed protein are indistinguishable from those of native bacteriorhodopsin (BR) in purple membrane of wild type strains. Strain Pho81BR permits for the first time characterization of effects of BR photoactivation alone on cell swimming behavior and energetics in the absence of the spectrally similar phototaxis receptor sensory rhodopsin I (SR-I) and electrogenic chloride pump halorhodopsin (HR). A non-adaptive upward shift in spontaneous swimming reversal frequency occurs following 3 s of continuous illumination of Pho81BR cells with green light (550 +/- 20 nm). This effect is abolished by low concentrations of the proton ionophore carbonylcyanide m-chlorophenylhydrazone. Although BR does not mediate phototaxis responses in energized Pho81BR cells under our culture conditions, proton pumping by BR in Pho81BR cells partially deenergized by inhibitors of respiration and adenosine triphosphate synthesis results in a small attractant response. Based on our measurements, we attribute the observed effects of BR photoactivation on swimming behavior to secondary consequences of electrogenic proton pumping on metabolic or signal transduction pathways, rather than to primary sensory signaling such as that mediated by SR-I. Proton extrusion by BR activates gated proton influx ports resulting in net proton uptake in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subpicosecond Excited-State Proton Transfer Preceding Isomerization During the Photorecovery of Photoactive Yellow Protein

The ultrafast excited-state dynamics underlying the receptor state photorecovery is resolved in the M100A mutant of the photoactive yellow protein (PYP) from Halorhodospira halophila. The M100A PYP mutant, with its distinctly slower photocycle than wt PYP, allows isolation of the pB signaling state for study of the photodynamics of the protonated chromophore cis-p-coumaric acid. Transient absorption signals indicate a subpicosecond excited-state proton-transfer reaction in the pB state that results in chromophore deprotonation prior to the cis-trans isomerization required in the photorecovery dynamics of the pG state. Two terminal photoproducts are observed, a blue-absorbing species presumed to be deprotonated trans-p-coumaric acid and an ultraviolet-absorbing protonated photoproduct. These two photoproducts are hypothesized to originate from an equilibrium of open and closed folded forms of the signaling state, I(2) and I(2)'.     

Protein structural dynamics of photoactive yellow protein in solution revealed by pump-probe X-ray solution scattering

Photoreceptor proteins play crucial roles in receiving light stimuli that give rise to the responses required for biological function. However, structural characterization of conformational transition of the photoreceptors has been elusive in their native aqueous environment, even for a prototype photoreceptor, photoactive yellow protein (PYP). We employ pump-probe X-ray solution scattering to probe the structural changes that occur during the photocycle of PYP in a wide time range from 3.16 μs to 300 ms. By the analysis of both kinetics and structures of the intermediates, the structural progression of the protein in the solution phase is vividly visualized. We identify four structurally distinct intermediates and their associated five time constants and reconstructed the molecular shapes of the four intermediates from time-independent, species-associated difference scattering curves. The reconstructed structures of the intermediates show the large conformational changes such as the protrusion of N-terminus, which is restricted in the crystalline phase due to the crystal contact and thus could not be clearly observed by X-ray crystallography. The protrusion of the N-terminus and the protein volume gradually increase with the progress of the photocycle and becomes maximal in the final intermediate, which is proposed to be the signaling state. The data not only reveal that a common kinetic mechanism is applicable to both the crystalline and the solution phases, but also provide direct evidence for how the sample environment influences structural dynamics and the reaction rates of the PYP photocycle.     

pH-dependent bending in and out of purple membranes comprising BR-D85T

The light-driven proton pump bacteriorhodopsin (BR) embedded in a purple membrane (PM) from Halobacterium salinarum undergoes a series of conformational changes while transporting a proton from the cytoplasmic to the extracellular side over the course of the so-called photocycle. Wild-type BR variant D85T, where aspartic acid 85 is replaced by threonine, allows for the study of structural intermediates of this photocycle that are formed in a light-dependent manner in the wild-type and in thermal equilibrium by tuning the pH of the D85T purple membrane suspension. Especially the last and least studied O-intermediate of the photocycle of bacteriorhodopsin has caught recent attention. First AFM images of D85T under acidic conditions resembling wild-type BR under physiological conditions in the O-photocycle-intermediate are presented. Bacteriorhodopsins embedded in the strongly bent purple membranes were analyzed by single molecule force spectroscopy (SMFS) providing the first single molecule force spectra of BR in the O-intermediate. SMFS was further employed to determine the absolute sign of membrane curvature. Complementary electrostatic force microscopy (EFM) was performed to support PM side discrimination and determination of the bending direction. Bending of PM-D85T was analyzed in more detail providing further insight into the structure-function relationship of the bacteriorhodopsin proton pump as well as PM behaviour at the solid-liquid junction. Findings reported here are of general interest to the field of chemomechanical transducers.     

S1P1-selective in vivo-active agonists from high-throughput screening: off-the-shelf chemical probes of receptor interactions, signaling, and fate

The essential role of the sphingosine 1-phosphate (S1P) receptor S1P(1) in regulating lymphocyte trafficking was demonstrated with the S1P(1)-selective nanomolar agonist, SEW2871. Despite its lack of charged headgroup, the tetraaromatic compound SEW2871 binds and activates S1P(1) through a combination of hydrophobic and ion-dipole interactions. Both S1P and SEW2871 activated ERK, Akt, and Rac signaling pathways and induced S1P(1) internalization and recycling, unlike FTY720-phosphate, which induces receptor degradation. Agonism with receptor recycling is sufficient for alteration of lymphocyte trafficking by S1P and SEW2871. S1P(1) modeling and mutagenesis studies revealed that residues binding the S1P headgroup are required for kinase activation by both S1P and SEW2871. Therefore, SEW2871 recapitulates the action of S1P in all the signaling pathways examined and overlaps in interactions with key headgroup binding receptor residues, presumably replacing salt-bridge interactions with ion-dipole interactions.     

The photoreaction of the photoactive yellow protein domain in the light sensor histidine kinase Ppr is influenced by the C-terminal domains

To study the role of the C-terminal domains in the photocycle of a light sensor histidine kinase (Ppr) having a photoactive yellow protein (PYP) domain as the photosensor domain, we analyzed the photocycles of the PYP domain of Ppr (Ppr-PYP) and full-length Ppr. The gene fragment for Ppr-PYP was expressed in Escherichia coli, and it was chemically reconstituted with p-coumaric acid; the full-length gene of Ppr was coexpressed with tyrosine ammonia-lyase and p-coumaric acid ligase for biosynthesis in cells. The light/dark difference spectra of Ppr-PYP were pH sensitive. They were represented as a linear combination of two independent difference spectra analogous to the PYP(L)/dark and PYP(M)/dark difference spectra of PYP from Halorhodospira halophila, suggesting that the pH dependence of the difference spectra is explained by the equilibrium shift between the PYP(L)- and PYP(M)-like intermediates. The light/dark difference spectrum of Ppr showed the equilibrium shift toward PYP(L) compared with that of Ppr-PYP. Kinetic measurements of the photocycles of Ppr and Ppr-PYP revealed that the C-terminal domains accelerate the recovery of the dark state. These observations suggest an interaction between the C-terminal domains and the PYP domain during the photocycle, by which light signals captured by the PYP domain are transferred to the C-terminal domains.     

Functional Expression of the Signaling Complex Sensory Rhodopsin II/Transducer II from Halobacterium salinarum in Escherichia coli†

Sensory rhodopsin II, a photoreceptor from Halobacterium salinarum (HsSRII), in complex with its cognate transducer protein (HsHtrII) triggers the photophobic response via a cytoplasmic two‐component signaling cascade. HsHtrII possess in addition to the HsSRII binding and the cytoplasmic domains an extracellular serine‐receptor domain. Here we describe the properties of HsSRII and HsHtrII and those of various shortened transducer analogs, heterologously expressed in Escherichia coli. HsSRII displays the photocycle typical of archaeal photosensors with prolonged kinetics. Using an isothermal titration calorimetric analysis for this complex a dissociation constant of 1.1 μm was obtained similar to that of the corresponding transducer/receptor pair from Natronobacterium pharaonis. A shortened transducer lacking the extracellular and cytoplasmic domain is also sufficient to bind the receptor with a slightly lower affinity. The dissociation constant of serine binding to the extracellular domain was determined to be about 5 μm . This result is in line with the proposal that the extracellular domain indeed is a serine receptor.     

Structures of the chromophore binding sites in BLUF domains as studied by molecular dynamics and quantum chemical calculations

BLUF (blue-light sensing using FAD) domains constitute a new family of flavin-based blue light photoreceptors. The photocycle of BLUF is unique in the sense that a few hydrogen bond rearrangements are accompanied by only slight structural changes in the bound chromophore. The hydrogen bond rearrangements upon illumination have been inferred from spectral changes in the chromophore: approximately 10 nm redshift of the absorption maximum and approximately 16 cm(-1) downshift of the C4=O stretching frequency. However, the exact features of the hydrogen bond network around the active site are still the subject of some controversy. In particular, the orientation of a conserved Gln (Gln63 in AppA) is presently one of the most questioned topics in the field. Here we perform molecular dynamics simulations for the wild-type AppA, AppA1-124C20S, BlrB and T110078 and furthermore quantum chemical calculations to investigate their spectroscopic properties in the dark and signaling states. On the basis of these results, we reveal the dynamic aspect of hydrogen bonding networks at the active site and propose theoretically reasonable models for the dark and signaling states of the BLUF domains.     

Dissecting the photocycle of the bacteriorhodopsin E204Q mutant from kinetic multichannel difference spectra. Extension of the method of singular value decomposition with self-modeling to five components.

Kinetic multichannel difference spectroscopy in the visible spectral range of the Glu204 --> Gln(E204Q) site-directed mutant of bacteriorhodopsin revealed five spectrally distinct metastable intermediates, as for the wild type. Due to the perturbation of the extracellular proton release cluster, the late O intermediate accumulates in much higher amounts in this mutant, and the photocycle is not complicated by the pH-dependent branching observed in the wild type protein. This mutant is therefore more amenable than the wild type to the determination of the intermediate spectra with the method of singular value decomposition with self-modeling, developed recently for three components (Zimányi et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4408-4413, 4414-4419). The method provides the most reliable spectra so far, defining the time evolution of the intermediates essential to the determination of the reaction scheme that describes the photocycle. The analysis confirms published results on this mutant by and large, but revises the locations of the L intermediates in the photocycle. In addition, it allows identification of the pH-dependent transitions of the photocycle, and offers an alternative mechanism for the pH dependence of the yield and kinetics of the late O intermediate.     

Photoisomerization and proton transfer in photoactive yellow protein

The photoactive yellow protein (PYP) is a bacterial photosensor containing a para-coumaryl thioester chromophore that absorbs blue light, initiating a photocycle involving a series of conformational changes. Here, we present computational studies to resolve uncertainties and controversies concerning the correspondence between atomic structures and spectroscopic measurements on early photocycle intermediates. The initial nanoseconds of the PYP photocycle are examined using time-dependent density functional theory (TDDFT) to calculate the energy profiles for chromophore photoisomerization and proton transfer, and to calculate excitation energies to identify photocycle intermediates. The calculated potential energy surface for photoisomerization matches key, experimentally determined, spectral parameters. The calculated excitation energy of the photocycle intermediate cryogenically trapped in a crystal structure by Genick et al. [Genick, U. K.; Soltis, S. M.; Kuhn, P.; Canestrelli, I. L.; Getzoff, E. D. Nature 1998, 392, 206-209] supports its assignment to the PYP(B) (I(0)) intermediate. Differences between the time-resolved room temperature (298 K) spectrum of the PYP(B) intermediate and its low temperature (77 K) absorbance are attributed to a predominantly deprotonated chromophore in the former and protonated chromophore in the latter. This contrasts with the widely held belief that chromophore protonation does not occur until after the PYP(L) (I(1) or pR) intermediate. The structure of the chromophore in the PYP(L) intermediate is determined computationally and shown to be deprotonated, in agreement with experiment. Calculations based on our PYP(B) and PYP(L) models lead to insights concerning the PYP(BL) intermediate, observed only at low temperature. The results suggest that the proton is more mobile between Glu46 and the chromophore than previously realized. The findings presented here provide an example of the insights that theoretical studies can contribute to a unified analysis of experimental structures and spectra.     

Thermodynamics of the early steps in the photocycle of Natronobacterium pharaonis halorhodopsin. Influence of medium and of anion substitution

The enthalpy (delta H) and structural volume changes (delta V) associated with the formation and decay of the early intermediate K600 in the photocycle of Natronobacterium pharaonis halorhodopsin (pHR), an inward-directed anion pump, were obtained by laser-induced optoacoustic spectroscopy. A large expansion is associated with K600 formation, its value depending on the medium and on the anion (Cl-, NO3-, Br-, I-). A smaller expansion is associated with K600 decay to L520. A contraction is found for the same step in the case of the azide-loaded pHR which is an efficient outward-directed proton pump. Thus, the conformational changes in L520 determine the direction and sign of charge translocation. The linear correlation between delta H and delta V for chloride-loaded pHR observed upon mild medium variations is attributed to enthalpy-entropy compensation effects and allows the calculation of the free-energy changes, delta GK = (97 +/- 16) kJ/mol and delta GKL = -(2 +/- 2) kJ/mol. Different from other systems, delta S correlates negatively with delta V in the first steps of the pHR photocycle. Thus, the space around the anion becomes larger and more rigid during each of these two steps. The photocycle quantum yield was 0.52 for chloride-pHR as measured by laser flash photolysis.     

Two Consecutive Polar Amino Acids at the End of Helix E are Important for Fast Turnover of the Archaerhodopsin Photocycle

Archaerhodopsins (ARs) is one of the members of microbial rhodopsins. Threonine 164 (T164) and serine 165 (S165) residues of the AR from Halorubrum sp. ejinoor (HeAR) are fully conserved in ARs, although they are far from the proton transfer channel and the retinal Schiff base, and are likely involved in a hydrogen‐bonding network at the end of the Helix E where most microbial rhodopsins assume a “bent structure”. In the present work, T164 and/or S165 were replaced with an alanine (A), and the photocycles of the mutants were analyzed with flash photolysis. The amino acid replacements caused profound changes to the photocycle of HeAR including prolonged photocycle, accelerated decay of M intermediate and appearance of additional two intermediates which were evident in T164A‐ and T164A/S165A‐HeAR photocyles. These results suggest that although T164 and S165 are located at the far end of the photoactive center, these two amino acid residues are important for maintaining the fast turnover of the HeAR photocycle. The underlying molecular mechanisms are discussed in relation to hydrogen‐bonding networks involving these two amino acids. Present study may arouse our interests to explore the functional role of the well‐conserved “bent structure” in different types of microbial rhodopsin.     

Coupling caspase cleavage and proteasomal degradation of proteins carrying PEST motif

The degradation is critical to activation and deactivation of regulatory proteins involved in signaling pathways to cell growth, differentiation, stress responses and physiological cell death. Proteins carry domains and sequence motifs that function as prerequisite for their proteolysis by either individual proteases or the 26S multicomplex proteasomes. Two models for entry of substrates into the proteasomes have been considered. In one model, it is proposed that the ubiquitin chain attached to the protein serves as recognition element to drag them into the 19S regulatory particle, which promotes the unfolding required to its access into the 20S catalytic chamber. In second model, it is proposed that an unstructured tail located at amino or carboxyl terminus directly track proteins into the 26S/20S proteasomes. Caspases are cysteinyl aspartate proteases that control diverse signaling pathways, promoting the cleavage at one or two sites of hundreds of structural and regulatory protein substrates. Caspase cleavage sites are commonly found within PEST motifs, which are segments rich in proline (P), glutamic acid (D), aspartic acid (E) and serine (S) or threonine (T) residues. Considering that N- and C- terminal peptide carrying PEST motifs form disordered loops in the globular proteins after caspase cleavage, it is postulated here that these exposed termini serve as unstructured initiation site, coupling caspase cleavage and ubiquitin-proteasome dependent and independent degradation of short-lived proteins. This could explain the inherent susceptibility to proteolysis among proteins containing PEST motif.     

STEADY HYPSOCHROMIC SHIFT OF PEAK RESPONSIVITY TO ATTRACTANT LIGHT FROM 590 nm TO 560 nm IN Halobacterium halobium*

Abstract— Peak responsivity of photoattraction in Halobacterium halobium cells shows steady hypsochromic shift from 590 nm wavelength under low irradiance conditions to 560 nm under high irradiance conditions. Inversion of the photoattractant response, as dependent on blue vs red background light, is compatible with the known properties of photochromic sensory rhodopsin-I (SR-I) with ground state maximum absorption at 587 nm. Relaxation of the photoattractant response in H. halobium, as a function of wavelength and irradiance, gives a hint at an antagonistic pigment or intermediate state, different from ground state SR-I, with peak sensitivity at 620 nm or even above. The less sensitive photoattractant response at 560 nm persists without photorelaxation and represents the peak responsivity under high irradiance conditions.     

Low-temperature spectroscopy of Met100Ala mutant of photoactive yellow protein

The trans-to-cis photoisomerization of the p-coumaroyl chromophore of photoactive yellow protein (PYP) triggers the photocycle. Met100, which is located in the vicinity of the chromophore, is a key residue for the cis-to-trans back-isomerization of the chromophore, which is a rate-determining reaction of the PYP photocycle. Here we characterized the photocycle of the Met100Ala mutant of PYP (M100A) by low temperature UV-visible spectroscopy. Irradiation of M100A at 80 K yielded a 380 nm species (M100A(BL)), while the corresponding intermediate of wild type (WT; PYP(BL)) is formed above 90 K. The amounts of redshifted intermediates produced from M100A (M100A(B') and M100A(L)) were substantially less than those from WT. While the near-UV intermediate (PYP(M)) is not formed from WT in glycerol samples at low temperature, M100A(M) was clearly observed above 190 K. These alterations of the photocycle of M100A were explained by the shift in the equilibrium between the intermediates. The carbonyl oxygen of the thioester linkage of the cis-chromophore in the photocycle intermediates is close to the phenyl ring of Phe96 (<3.5 A), which would be displaced by the mutation of Met100. These findings imply that the interaction between chromophore and amino acid residues near Met100 is altered during the early stage of the PYP photocycle.     

Direct and Indirect Evolution of Photoluminescent Semiconductor CdS Magic-Size Clusters through Their Precursor Compounds

Colloidal semiconductor II–VI metal chalcogenide (ME) magic-size clusters (MSCs) exhibit either an optical absorption singlet or doublet. In the latter case, a sharp photoluminescence (PL) signal is observed. Whether the PL-inactive MSCs transform to the PL-active ones is unknown. We show that PL-inactive CdS MSC-322 transforms to PL-active CdS MSC-328 and MSC-373 in the presence of acetic acid (HOAc). MSC-322 displays a sharp absorption at ≈322 nm, whereas MSC-328 and MSC-373 both have broad absorptions respectively around 328 and 373 nm. In a reaction of cadmium myristate and S powder in 1-octadecene, MSC-322 develops; with HOAc, MSC-328 and MSC-373 are present. We propose that the MSCs evolve from their relatively transparent precursor compounds (PCs). The PC-322 to PC-328 quasi-isomerization involves monomer substitution, while monomer addition occurs for the PC-328 to PC-373 transformation. Our findings suggest that S dominates the precursor self-assembly quantitatively, and ligand-bonded Cd mainly controls MSC optical properties.     

SMM-chemokines: a class of unnatural synthetic molecules as chemical probes of chemokine receptor biology and leads for therapeutic development

Chemokines and their receptors play important roles in numerous physiological and pathological processes. To develop natural chemokines into receptor probes and inhibitors of pathological processes, the lack of chemokine-receptor selectivity must be overcome. Here, we apply chemical synthesis and the concept of modular modifications to generate unnatural synthetically and modularly modified (SMM)-chemokines that have high receptor selectivity and affinity, and reduced toxicity. A proof of the concept was shown by transforming the nonselective viral macrophage inflammatory protein-II into new analogs with enhanced selectivity and potency for CXCR4 or CCR5, two principal coreceptors for human immunodeficiency virus (HIV)-1 entry. These new analogs provided insights into receptor binding and signaling mechanisms and acted as potent HIV-1 inhibitors. These results support the concept of SMM-chemokines for studying and controlling the function of other chemokine receptors.     

Biosynthetic approach to modeling and understanding metalloproteins using unnatural amino acids

Metalloproteins have inspired chemists for many years to synthesize artificial catalysts that mimic native enzymes.As a complementary approach to studying native enzymes or making synthetic models,biosynthetic approach using small and stable proteins to model native enzymes has offered advantages of incorporating non-covalent secondary sphere interactions under physiological conditions.However,most biosynthetic models are restricted to natural amino acids.To overcome this limitation,incorporating unnatural amino acids into the biosynthetic models has shown promises.In this review,we summarize first synthetic,semisynthetic and biological methods of incorporates unnatural amino acids(UAAs)into proteins,followed by progress made in incorporating UAAs into both native metalloproteins and their biosynthetic models to fine-tune functional properties beyond native enzymes or their variants containing natural amino acids,such as reduction potentials of azurin,O_2 reduction rates and percentages of product formation of HCO models in Mb,the rate of radical transport in ribonucleotide reductase(RNR)and the proton and electron transfer pathways in photosystemⅡ(PSⅡ).We also discuss how this endeavour has allowed systematic investigations of precise roles of conserved residues in metalloproteins,such as Metl21 in azurin,Tyr244 that is cross-linked to one of the three His ligands to CuB in HCO,Tyr122,356,730 and 731 in RNR and TyrZ in PSⅡ.These examples have demonstrated that incorporating UAAs has provided a new dimension in our efforts to mimic native enzymes and in providing deeper insights into structural features responsible high enzymatic activity and reaction mechanisms,making it possible to design highly efficient artificial catalysts with similar or even higher activity than native enzymes.