QUATERNARY STRUCTURE OF PEA PHYTOCHROME I DIMER STUDIED WITH SMALL-ANGLE X-RAY SCATTERING and ROTARY-SHADOWING ELECTRON MICROSCOPY

Abstract— The quaternary structure of pea phytochrome type I (PI) dimer in the red-light-absorbing form was studied by small-angle X-ray scattering (SAXS) technique and rotary-shadowing electron microscopy. Structural parameters for PI 114 kDa chromopeptide dimer and its tryptically digested N-terminal 59 kDa chromopeptide monomer, such as average electron density, molecular volume and the second moment of electron density distribution, were determined in terms of SAXS using the contrast variation method. Furthermore, by means of model simulation for the scattering profiles of the chromopeptides, most plausible structural models for both peptides were constructed. The distance between the chromophoric domains was estimated to be about 70 A in the resultant model for 114 kDa chromopeptide dimer. Furthermore, the model was consistent with the electron-micrographic images of both the intact PI dimer and the PI 114 kDa chromopeptide dimer, so that the N-terminal 7 kDa fragment did not significantly contribute the low-resolution images of the dimer.     

Primary processes during the light-signal transduction of phototropin

Phototropin is a blue-light photoreceptor in plants that mediates phototropism, chloroplast relocation, stomata opening and leaf expansion. Phototropin molecule has two photoreceptive domains named LOV1 (light-oxygen-voltage) and LOV2 in the N-terminus and a serine/threonine kinase domain in the C-terminus, and acts as a blue light-regulated kinase. Each LOV domain binds a flavin mononucleotide as a chromophore and undergoes unique cyclic reactions upon blue-light absorption that comprises a cysteinyl-flavin adduct formation through a triplet-excited state and a successive adduct break to revert to the initial ground state. The molecular reactions underlying the photocycle are reviewed and one of the probable molecular schemes is presented. Adduct formation alters the secondary protein structure of the LOV domains. This structural change could be transferred to the linker between the kinase domain and involved in the photoregulation of the kinase activity. The structural changes as well as the oligomeric structures seem to differ between LOV1 and LOV2, which may explain the proposed roles of each domain in the photoregulation of the kinase activity. The photoregulation mechanism of phototropin kinase is reviewed and discussed in reference to the regulation mechanism of protein kinase A, which it resembles.     

FORMATION OF AGGREGATES OF TRYPTIC FRAGMENTS DERIVED FROM THE CARBOXYL- TERMINAL HALF OF PEA PHYTOCHROME and LOCALIZATION OF THE SITE OF CONTACT BETWEEN THE FRAGMENTS BY AMINO-TERMINAL AMINO ACID SEQUENCE ANALYSIS*

Limited trypsinization of large pea (Pisum sativum cv. Alaska) phytochrome and subsequent size-exclusion chromatography (SEC) in 0.1 M Na phosphate, pH 7.8, yielded a high-molecular-mass aggregate of tryptic fragments of phytochrome. Further SEC in 0.1 M Tris-HCl, pH 7.5, plus various concentrations of NaSCN, indicated that the tryptic-fragment complex contained an aggregate of 7 fragments of molecular mass from 38 to 55 kDa. The amino-terminal sequence of each fragment was determined from the samples electroblotted from sodium dodecylsulfate polyacrylamide gels onto polyvinylidene difluoride membranes, in order to localize the various fragments on the phytochrome polypeptide chain. All of the 7 fragments in the aggregate were found to be derived from the carboxyl-terminal half of phytochrome. A portion of the polypeptide chain (from Ala-752 to Arg-1000) common to all the tryptic fragments has been assigned as the site(s) of contact of the fragments. The tryptic-fragment complex, as well as large phytochrome itself, has been shown by SEC to dissociate to monomers in 2 M NaSCN. The result indicates that the main force involved in maintaining the complex and in contacts between monomers of phytochrome is non-ionic in nature. Relationship between the contact site(s) of the tryptic-fragment complex and large phytochrome monomer is discussed.     

A MODEL FOR THE MOLECULAR STRUCTURE AND ORIENTATION OF THE CHROMOPHORE IN A DIMERIC PHYTOCHROME MOLECULE*

A model for the molecular structure and orientation of red-light absorbing form of phytochrome (P,) chromophores in a dimeric molecular model of P_r is proposed. A chromophore model with probable molecular structures was generated to reproduce the absorption spectrum produced by its π-electron conjugating system. The model has C₅-Z, syn, C₁₀-E, anti and C₁₅-Z, syn configurations and a protonation at a C-ring nitrogen. Orientation of the chromophore model in the dimeric phytochrome molecular was analyzed by displaying the atoms of the chromophore, the coordinates of which were converted into those with respect to the molecular axes to the dimeric molecule, on a 3-D graphic workstation. The conversions were performed by using the azimuthal angles between the Z axis of the dimeric molecule (axis of 2-fold rotational symmetry) and the dipole moments of the electronic transition at the blue- (384 nm) and red- (667 nm) absorbing bands of the chromophore, which were calculated as 55.5° and 59.3°, respectively, based on linear dichroism of the oriented phytochrome molecules. The result demonstrates that the long axis of the P, chromophore lies almost parallel to the Y axis of the molecular model, and that the tetrapyrrolic chromophore is well contained within the flat chromophoric domain without protruding from it, a configuration that assures that the chromophore is protected against aqueous environments. The model may explain the rotation angle of the transition moment of the red-absorbing band, induced by the phototransformation from P_r to P_rr which we measured as smaller than that measured in nonoriented preparations by a photoselection technique. The model also suggests a molecular basis for the polarotropic response of phytochrome.     

Reactive cysteine is protonated in the triplet excited state of the LOV2 domain in Adiantum phytochrome3

Phototropin is a plant blue-light sensor protein that possesses a flavin mononucleotide (FMN) as the chromophore in LOV domains. Its photoreaction is an adduct formation between FMN and a nearby cysteine that takes place in the triplet excited state of FMN. In this communication, we revealed that the reactive cysteine is protonated in the triplet excited state of the LOV2 domain of Adiantum phytochrome3 by means of low-temperature FTIR spectroscopy. Its hydrogen-bonding interaction is strengthened in the triplet excited state, presumably with the FMN chromophore. Such strong interaction drives adduct formation on a microsecond time scale.     

PHOTOREACTIVATION OF Halobacterium halobium: ACTION SPECTRUM AND ROLE OF PIGMENTATION

Abstract— An action spectrum for photoreactivation was measured with Halobacterium halobium R₁m₁ to prove a role of carotenoid pigments in photoreactivation of the bacteria. The action spectrum obtained showed a main peak at 435 nm and a minor peak at about 325 nm. The action spectrum was similar to that of Streptomyces pigment (Eker et al. , 1981) suggesting that the chromophore of the photoreactivating enzyme in Halobacterium halobium is 8-OH-5-deazaflavin. The minor peak may be due to photochemical cleavage of a pyrimidine6–4 hetero adduct. The result indicates that carotenoid pigments do not play a positive role in enhancing photoreactivation. This was confirmed also by comparing the efficiency of photoreactivation at 465 nm among three strains of Halobacterium halobium having different carotenoid pigments; R₁m₁. R₁ and W5002–1.     

Macromolecular crowding effects on reactions of TePixD (Tll0078)

To reveal macromolecular crowding effects on a chemical reaction of a BLUF (sensors of blue light using FAD) protein (PixD from a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 [TePixD, Tll0078]), the photoreaction was studied at various concentrations of the macromolecule Ficoll-70 by UV/Vis absorption spectroscopy and the pulsed laser-induced transient grating (TG) method. The absorption spectrum did not change with varying concentration of Ficoll-70. The crowding did not affect the quantum yield of the spectral red shift reaction, recovery rate of the product, rate constant of the volume change reaction and the magnitude of the volume change. However, the magnitude of the TG signal representing the diffusion-sensitive conformation change significantly increased on addition of Ficoll-70. This dependence was attributed to the crowding effect on the TePixD decamer-pentamer equilibrium in the solution. This result indicates that the TePixD reaction is more efficient in cellular than in in vitro conditions.     

FLASH-INDUCED FAST CHANGE IN SURFACE POTENTIAL AND FLUIDITY OF PURPLE MEMBRANE STUDIED BY SPIN LABEL METHOD

Abstract— Flash-induced changes in surface potential and fluidity of purple membrane were studied by a spin label method. Changes in surface potential and fluidity were monitored by observing the distribution of charged and uncharged spin labels between the purple membrane and the aqueous phase.
On flash illumination, a transient hyperpolarization of the surface potential and a transient fluidization of the membrane hydrophobic region are induced. The former may probably reflect the proton movement near the purple membrane surface, while the latter may result from photo-induced conformational changes of bacteriorhodopsin.
The two events are different in time course. The relationships between the two events, and the formation and decay of the intermediates of bacteriorhodopsin in the photoreaction cycle are discussed.     

PHOTOREVERSIBLE CHANGES IN pH OF PEA PHYTOCHROME SOLUTIONS

Abstract— Photoinduced pH changes in unbuffered solutions of undegraded pea phytochrome were studied at 10_oC by using a glass electrode. Red light irradiation caused alkalinization of the solutions in the pH range 5.2–xs7.5 and acidification in pH 7.5–8.9. The pH changes were fully reversed by a subsequent irradiation with far-red light. The red and far-red light effects were repeatedly reversible. The solution of tryptic peptide of phytochrome (mol. wt 60000) showed similar photoreversible pH changes.     

Photoreaction of the cysteine S-H group in the LOV2 domain of Adiantum phytochrome3

Phy3-LOV2 is the chromophore domain of a blue-light photoreceptor for tropic responses of plants. FMN is noncovalently bound to phy3-LOV2, and the protein structure is characteristic of the LOV (light-oxygen-voltage) domain. Primary photoreaction is considered to be adduct formation between FMN and a cysteine, while deprotonation of the cysteine S-H group was suggested. On the basis of the infrared spectral analysis, however, we have shown that the cysteine of phy3-LOV2 is in the protonated S-H form, and not in the thiolate form in the ground state. Upon formation of S390, the S-H group disappears, presumably forming the adduct between FMN and Cys966. S390 can be trapped at 150 K, and the protein structure of S390 may not be changed drastically at 295 K.