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991.
Andrew T. Ulijasz Gabriel Cornilescu David von Stetten Claudia Cornilescu Francisco Velazquez Escobar Junrui Zhang Robert J. Stankey Mario Rivera Peter Hildebrandt Richard D. Vierstra 《The Journal of biological chemistry》2009,284(43):29757-29772
Phytochromes are a collection of bilin-containing photoreceptors that regulate a diverse array of processes in microorganisms and plants through photoconversion between two stable states, a red light-absorbing Pr form, and a far red light-absorbing Pfr form. Recently, a novel set of phytochrome-like chromoproteins was discovered in cyanobacteria, designated here as cyanochromes, that instead photoconvert between stable blue and green light-absorbing forms Pb and Pg, respectively. Here, we show that the distinctive absorption properties of cyanochromes are facilitated through the binding of phycocyanobilin via two stable cysteine-based thioether linkages within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. Absorption, resonance Raman and infrared spectroscopy, and molecular modeling of the Te-PixJ GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA) domain assembled with phycocyanobilin are consistent with attachments to the C31 carbon of the ethylidene side chain and the C4 or C5 carbons in the A–B methine bridge to generate a double thioether-linked phycoviolobilin-type chromophore. These spectroscopic methods combined with NMR data show that the bilin is fully protonated in the Pb and Pg states and that numerous conformation changes occur during Pb → Pg photoconversion. Also identified were a number of photochromically inactive mutants with strong yellow or red fluorescence that may be useful for fluorescence-based cell biological assays. Phylogenetic analyses detected cyanochromes capable of different signaling outputs in a wide range of cyanobacterial species. One unusual case is the Synechocystis cyanochrome Etr1 that also binds ethylene, suggesting that it works as a hybrid receptor to simultaneously integrate light and hormone signals.Phytochromes (Phys)3 comprise a large and diverse superfamily of photoreceptors that regulate a wide range of physiological responses in plants, fungi, bacteria, and cyanobacteria (1–3). They are unique among photoreceptors by being able to photoconvert between two stable states, a red light-absorbing Pr form that is typically the dark-adapted and biologically inactive conformer and a far-red light-absorbing Pfr form that requires light for its production and is typically the biologically active conformer. By interconverting between Pr and Pfr, Phys act as light-regulated switches in controlling processes ranging from phototaxis and pigmentation in bacteria to seed germination, photomorphogenesis, and flowering time in higher plants.Light absorption by Phys is directed by a bilin (or linear tetrapyrrole) chromophore produced by the oxidative cleavage of heme. Although bacterial and fungal Phys use the immediate cleavage product biliverdin (BV), cyanobacterial and higher plant Phys use phycocyanobilin (PCB) and phytochromobilin, respectively, produced by enzymatic reduction of BV (1, 2). The bilin is then covalently bound autocatalytically to the photosensory unit of the apoprotein, which typically contains a sequence of Per/Arndt/Sim (PAS), cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF), and Phy-associated (PHY) domains. Intimate contact between the bilin and surrounding protein residues then generates the unique photochromic properties of Phys. Recent three-dimensional structures of the Pr form of several bacterial Phys (BphPs) and two cyanobacterial Phys (Cphs) have shown that the bilin is deeply buried within the GAF domain in a ZZZssa configuration and that the connection between the GAF and PAS domains is stabilized by a rare figure-of-eight knot involving the region upstream of the PAS domain being lassoed by a conserved loop within the GAF domain (4–9). Although the structure of Pfr remains unsolved, various physicochemical studies have proposed that photoconversion involves a rotation of one of the three methine bridges between the pyrrole rings (1, 10–14). This rotation then induces much slower thermally driven movements of the protein to initiate signal transduction.In microorganisms, Pfr can activate a variety of signaling systems using output motifs directly appended to the C-terminal end of the photosensory region. The most prevalent are histidine kinase domains that then begin specific two-component phosphorelays (3, 15, 16). Although the output of plant Phys remains unclear, the presence of a C-terminal HK-related domain suggests that they also work as light-regulated protein kinases (17).In addition to the canonical Phys, it has become apparent through phylogenetic and biochemical studies that a heterogeneous collection of Phy-like photoreceptors exists (e.g. Refs. 3 and 18). These include Phys that prefer Pfr as the dark-adapted state (7, 19, 20), Phys that photoconvert from Pr to shorter wavelength-absorbing “near red” or Pnr forms (6, 21), and Phy-like photoreceptors that bind bilins but instead photoconvert between forms with maximal absorption other than red and far-red light (22–25). Often these Phy-like sequences are missing key residues or domains common among canonical Phys, suggesting that they employ novel bilins as chromophores, bind the bilin in different architectures, and/or use distinct photochemistries.One subclass of novel Phy-like photoreceptors present in a number of cyanobacteria, which we have designated cyanochromes (or Cycs) to better distinguish them from Cphs, is exemplified by Synechocystis sp. PCC6803 (Syn) PixJ (or TaxD1, locus sll0041) and its relatives. Syn-PixJ was discovered based on its involvement in blue light-mediated phototaxis in this mesophilic cyanobacterium (26, 27) with its close homolog Te-PixJ (locus tll0569) then found in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 by sequence similarity (28). Like Cphs, the cyanochromes tested thus far covalently bind PCB but then generate photoreceptors that convert between blue and green light-absorbing forms designated Pb and Pg, respectively (22, 24, 29). Subsequent studies proposed that PCB is converted to phycoviolobilin (PVB) upon attachment to the apoprotein (30). PVB differs from PCB by having a methylene instead of a methine bridge between the A and B pyrrole rings, which blue-shifts the absorption of the chromophore by shortening the π-conjugation system. Phototransformation of Pb to Pg could then occur by a mechanism similar to Phys.How Te-PixJ and related cyanochromes bind PCB to generate more blue-shifted PVB-type chromophores remains unclear. Like Cphs, two cyanochromes examples link PCB via a thioether linkage between a cysteine in the Cyc-GAF domain and the C31 carbon of the ethylidene side chain of ring A (24, 28). Additionally, loss of the C4C5 double bond is necessary to generate PVB. One model by Ishizuka et al. (30) from studies with Te-PixJ proposed that the double bond moves from the C4-C5 position to the C2-C3 position by an autoisomerase activity intrinsic to the GAF domain. A more recent model by Rockwell et al. (24) using another Syn-PixJ relative in T. elongatus, Tlr0924, invoked the possibility of a second cysteine that also participates in PCB ligation. This cysteine was proposed to bind the bilin at the C10 position via a reversible thioether linkage. In the dark-adapted Pb state, the second linkage would then be formed to generate a rubin-like chromophore attached to the bridge between the B and C pyrrole rings. This bond would then break upon photoconversion to generate the more π-conjugated green light-absorbing photoproduct Pg.In this report, we employed a number of physicochemical approaches to help resolve the unique chromophore architecture and photochemical properties of cyanochromes, using Te-PixJ as the example. By independently mutagenizing the cysteine that binds the A ring ethylidene (Cys-522 (22)) and that proposed by Rockwell et al. (24) to reversibly bind the bilin at a second site (Cys-494), we demonstrate that both residues form light-stable covalent adducts with a PVB-type chromophore. In addition, we employed various spectroscopic methods to show that the bound PVB is fully protonated as both Pb and Pg, that only one pyrrole ring is active during photoconversion, and that the polypeptide may undergo extensive remodeling as Pb converts to Pg. We identified a set of conserved amino acids in Te-PixJ important for cyanochrome photochemistry, including several that when substituted generate yellow or red fluorescent chromoproteins potentially useful for cell biological applications. Phylogenetic analyses show that cyanochromes are widespread among cyanobacteria with their closest relatives being members of the red/far-red light-absorbing Phy subfamily defined by the absence of the N-terminal PAS domain (31). 相似文献
992.
993.
Andrew J.Parsons Ifty Ahmed Papia Haque Ben Fitzpatrick Muhammad I.K.Niazi Gavin S.Walker Chris D.Rudd 《仿生工程学报(英文版)》2009,6(4):318-323
We investigate high-modulus degradable materials intended to replace metals in biomedical applications.These are typicallycomposites comprising a polylactide(PLA)matrix reinforced with phosphate glass fibres,which provide reinforcementsimilar to E-glass but are entirely degradable in water to produce,principally,calcium phosphate.We have made compositesusing a variety of fibre architectures,from non-woven random mats to unidirectional fibre tapes.Flexural properties in theregion of 30 GPa modulus and 350 MPa strength have been achieved-directly comparable to quoted values for human corticalbone.In collaboration with other groups we have begun to consider the development of foamed systems with structures mimickingcancellous bone and this has shown significant promise.The fibres in these foamed structures provide improved creepresistance and reinforcement of the pore walls.To date the materials have exhibited excellent cellular responses in vitro andfurther studies are due to include consideration of the surface character of the materials and the influence of this on cell interaction,both with the composites and the glass fibres themselves,which show promise as a standalone porous scaffold. 相似文献
994.
Molecular sensors based on intramolecular Förster resonance energy transfer (FRET) have become versatile tools to monitor regulatory molecules in living tissue. However, their use is often compromised by low signal strength and excessive noise. We analyzed signal/noise (SNR) aspects of spectral FRET analysis methods, with the following conclusions: The most commonly used method (measurement of the emission ratio after a single short wavelength excitation) is optimal in terms of signal/noise, if only relative changes of this uncalibrated ratio are of interest. In the case that quantitative data on FRET efficiencies are required, these can be calculated from the emission ratio and some calibration parameters, but at reduced SNR. Lux-FRET, a recently described method for spectral analysis of FRET data, allows one to do so in three different ways, each based on a ratio of two out of three measured fluorescence signals (the donor and acceptor signal during a short-wavelength excitation and the acceptor signal during long wavelength excitation). Lux-FRET also allows for calculation of the total abundance of donor and acceptor fluorophores. The SNR for all these quantities is lower than that of the plain emission ratio due to unfavorable error propagation. However, if ligand concentration is calculated either from lux-FRET values or else, after its calibration, from the emission ratio, SNR for both analysis modes is very similar. Likewise, SNR values are similar, if the noise of these quantities is related to the expected dynamic range. We demonstrate these relationships based on data from an Epac-based cAMP sensor and discuss how the SNR changes with the FRET efficiency and the number of photons collected. 相似文献
995.
Pacific halibut collected in the Aleutian Islands, Bering Sea and Gulf of Alaska were used to test the hypothesis of genetic
panmixia for this species in Alaskan marine waters. Nine microsatellite loci and sequence data from the mitochondrial (mtDNA)
control region were analyzed. Eighteen unique mtDNA haplotypes were found with no evidence of geographic population structure.
Using nine microsatellite loci, significant heterogeneity was detected between Aleutian Island Pacific halibut and fish from
the other two regions (F
ST range = 0.007–0.008). Significant F
ST values represent the first genetic evidence of divergent groups of halibut in the central and western Aleutian Archipelago.
No significant genetic differences were found between Pacific halibut in the Gulf of Alaska and the Bering Sea leading to
questions about factors contributing to separation of Aleutian halibut. Previous studies have reported Aleutian oceanographic
conditions at deep inter-island passes leading to ecological discontinuity and unique community structure east and west of
Aleutian passes. Aleutian Pacific halibut genetic structure may result from oceanographic transport mechanisms acting as partial
barriers to gene flow with fish from other Alaskan waters. 相似文献
996.
Mosyak L Wood A Dwyer B Buddha M Johnson M Aulabaugh A Zhong X Presman E Benard S Kelleher K Wilhelm J Stahl ML Kriz R Gao Y Cao Z Ling HP Pangalos MN Walsh FS Somers WS 《The Journal of biological chemistry》2006,281(47):36378-36390
Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200 A2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane. 相似文献
997.
Paterson AH Chapman BA Kissinger JC Bowers JE Feltus FA Estill JC 《Trends in genetics : TIG》2006,22(11):597-602
Genome duplication is potentially a good source of new genes, but such genes take time to evolve. We have found a group of "duplication-resistant" genes, which have undergone convergent restoration to singleton status following several independent genome duplications. Restoration of duplication-resistant genes to singleton status could be important to long-term survival of a polyploid lineage. Angiosperms show more frequent polyploidization and a higher degree of duplicate gene preservation than other paleopolyploids, making them well-suited to further study of duplication-resistant genes. 相似文献
998.
999.
A deletion at the mouse Xist gene exposes trans-effects that alter the heterochromatin of the inactive X chromosome and the replication time and DNA stability of both X chromosomes 下载免费PDF全文
Diaz-Perez SV Ferguson DO Wang C Csankovszki G Wang C Tsai SC Dutta D Perez V Kim S Eller CD Salstrom J Ouyang Y Teitell MA Kaltenboeck B Chess A Huang S Marahrens Y 《Genetics》2006,174(3):1115-1133
The inactive X chromosome of female mammals displays several properties of heterochromatin including late replication, histone H4 hypoacetylation, histone H3 hypomethylation at lysine-4, and methylated CpG islands. We show that cre-Lox-mediated excision of 21 kb from both Xist alleles in female mouse fibroblasts led to the appearance of two histone modifications throughout the inactive X chromosome usually associated with euchromatin: histone H4 acetylation and histone H3 lysine-4 methylation. Despite these euchromatic properties, the inactive X chromosome was replicated even later in S phase than in wild-type female cells. Homozygosity for the deletion also caused regions of the active X chromosome that are associated with very high concentrations of LINE-1 elements to be replicated very late in S phase. Extreme late replication is a property of fragile sites and the 21-kb deletions destabilized the DNA of both X chromosomes, leading to deletions and translocations. This was accompanied by the phosphorylation of p53 at serine-15, an event that occurs in response to DNA damage, and the accumulation of gamma-H2AX, a histone involved in DNA repair, on the X chromosome. The Xist locus therefore maintains the DNA stability of both X chromosomes. 相似文献
1000.
In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells 总被引:3,自引:0,他引:3
Mumm JS Williams PR Godinho L Koerber A Pittman AJ Roeser T Chien CB Baier H Wong RO 《Neuron》2006,52(4):609-621
Targeting of axons and dendrites to particular synaptic laminae is an important mechanism by which precise patterns of neuronal connectivity are established. Although axons target specific laminae during development, dendritic lamination has been thought to occur largely by pruning of inappropriately placed arbors. We discovered by in vivo time-lapse imaging that retinal ganglion cell (RGC) dendrites in zebrafish show growth patterns implicating dendritic targeting as a mechanism for contacting appropriate synaptic partners. Populations of RGCs labeled in transgenic animals establish distinct dendritic strata sequentially, predominantly from the inner to outer retina. Imaging individual cells over successive days confirmed that multistratified RGCs generate strata sequentially, each arbor elaborating within a specific lamina. Simultaneous imaging of RGCs and subpopulations of presynaptic amacrine interneurons revealed that RGC dendrites appear to target amacrine plexuses that had already laminated. Dendritic targeting of prepatterned afferents may thus be a novel mechanism for establishing proper synaptic connectivity. 相似文献