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1.
Sung Hyun Kim So Young Kim Kwang-Hwan Jung 《Bioscience, biotechnology, and biochemistry》2013,77(7):1070-1074
Anabaena sensory rhodopsin transducer (ASRT) is believed to be a major player in the photo-signal transduction cascade, which is triggered by Anabaena sensory rhodopsin. Here, we characterized DNA binding activity of ASRT probed by using fluorescence correlation spectroscopy. We observed clear decrease of diffusion coefficient of DNA upon binding of ASRT. The dissociation constant, KD, of ASRT to 20?bp-long DNA fragments lied in micro-molar range and varied moderately with DNA sequence. Our results suggest that ASRT may interact with several different regions of DNA with different binding affinity for global regulation of several genes that need to be activated depending on the light illumination. 相似文献
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So Young Kim Sa Ryong Yoon SongI Han Yuna Yun Kwang‐Hwan Jung 《Molecular microbiology》2014,93(3):403-414
In 2003, Anabaena sensory rhodopsin (ASR), a membrane‐bound light sensor protein, was discovered in cyanobacteria. Since then, a large number of functions have been described for ASR, based on protein biochemical and biophysical studies. However, no study has determined the in vivo mechanism of photosensory transduction for ASR and its transducer protein (ASRT). Here, we aimed to determine the role of ASRT in physiological photo‐regulation. ASRT is known to be related to photochromism, because it regulates the expression of phycocyanin (cpc‐gene) and phycoerythrocyanin (pec gene), two major proteins of the phycobilisome in cyanobacteria. By examining wild type and knockout mutant Anabaena cells, we showed that ASRT repressed the expression of these two genes. We also demonstrated physical interactions between ASRT, ASR, and the promoter regions of cpc, pec, kaiABC (circadian clock gene) and the asr operon, both in vitro and in vivo. Binding assays indicated that ASRT had different sites of interaction for binding to ASR and DNA promoter regions. ASRT also influenced the retinal re‐isomerization rate in dark through a physical interaction with ASR, and it regulated reporter gene expression in vivo. These results suggested that ASRT relayed the photosignal from ASR and directly regulated gene expression. 相似文献
4.
Solid-state NMR spectroscopy is an efficient tool for following conformational dynamics of membrane proteins at atomic resolution. We used this technique for the site-specific detection of light-induced hydrogen-deuterium exchange in the lipid-embedded heptahelical transmembrane photosensor Anabaena sensory rhodopsin to pinpoint the location of its conformational changes upon activation. We show that the light-induced conformational changes result in a dramatic, but localized, increase in the exchange in the transmembrane regions. Most notably, the cytoplasmic half of helix G and the cytoplasmic ends of helices B and C exchange more extensively, probably as a result of their relative displacement in the activated state, allowing water to penetrate into the core of the protein. These light-induced rearrangements must provide the structural basis for the photosensory function of Anabaena sensory rhodopsin. 相似文献
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Suttisrisung S Senapin S Withyachumnarnkul B Wongprasert K 《Journal of biosciences》2011,36(5):833-843
A legume-type lectin (L-lectin) gene of the red algae Gracilaria fisheri (GFL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of GFL was 1714 bp and contained a 1542 bp
open reading frame encoding 513 amino acids with a predicted molecular mass of 56.5 kDa. Analysis of the putative amino acid
sequence with NCBI-BLAST revealed a high homology (30–68%) with legume-type lectins (L-lectin) from Griffithsia japonica, Clavispora lusitaniae, Acyrthosiphon pisum, Tetraodon nigroviridis and Xenopus tropicalis. Phylogenetic relationship analysis showed the highest sequence identity to a glycoprotein of the red algae Griffithsia japonica (68%) (GenBank number AAM93989). Conserved Domain Database analysis detected an N-terminal carbohydrate recognition domain
(CRD), the characteristic of L-lectins, which contained two sugar binding sites and a metal binding site. The secondary structure
prediction of GFL showed a β-sheet structure, connected with turn and coil. The most abundant structural element of GFL was
the random coil, while the α-helixes were distributed at the N- and C-termini, and 21 β-sheets were distributed in the CRD.
Computer analysis of three-dimensional structure showed a common feature of L-lectins of GFL, which included an overall globular
shape that was composed of a β-sandwich of two anti-parallel β-sheets, monosaccharide binding sites, were on the top of the
structure and in proximity with a metal binding site. Northern blot analysis using a DIG-labelled probe derived from a partial
GFL sequence revealed a hybridization signal of ~1.7 kb consistent with the length of the full-length GFL cDNA identified
by RACE. No detectable band was observed from control total RNA extracted from filamentous green algae. 相似文献
7.
Vogeley L Trivedi VD Sineshchekov OA Spudich EN Spudich JL Luecke H 《Journal of molecular biology》2007,367(3):741-751
We present crystal structures of the Anabaena sensory rhodopsin transducer (ASRT), a soluble cytoplasmic protein that interacts with the first structurally characterized eubacterial retinylidene photoreceptor Anabaena sensory rhodopsin (ASR). Four crystal structures of ASRT from three different spacegroups were obtained, in all of which ASRT is present as a planar (C4) tetramer, consistent with our characterization of ASRT as a tetramer in solution. The ASRT tetramer is tightly packed, with large interfaces where the well-structured beta-sandwich portion of the monomers provides the bulk of the tetramer-forming interactions, and forms a flat, stable surface on one side of the tetramer (the beta-face). Only one of our four different ASRT crystals reveals a C-terminal alpha-helix in the otherwise all-beta protein, together with a large loop from each monomer on the opposite face of the tetramer (the alpha-face), which is flexible and largely disordered in the other three crystal forms. Gel-filtration chromatography demonstrated that ASRT forms stable tetramers in solution and isothermal microcalorimetry showed that the ASRT tetramer binds to ASR with a stoichiometry of one ASRT tetramer per one ASR photoreceptor with a K(d) of 8 microM in the highest affinity measurements. Possible mechanisms for the interaction of this transducer tetramer with the ASR photoreceptor via its flexible alpha-face to mediate transduction of the light signal are discussed. 相似文献
8.
Julia Marín-Navarro Leontina Gurgu Santiago Alamar Julio Polaina 《Applied microbiology and biotechnology》2011,89(1):121-130
Saccharomyces cerevisiae Sta1 glucoamylase and Saccharomycopsis fibuligera Bgl1 β-glucosidase, two relevant enzymes from a biotechnological point of view, are proteins with multidomain structure.
Starting with homology-based structural models of Sta1 and Bgl1, we have constructed a series of hybrid enzymes by interchanging
domains of the two proteins. The first purpose of these constructs was to check available hypotheses about the uncertain biological
functions of two domains: the serine/threonine-rich domain (STRD) of Sta1 and a β-sandwich domain present in Bgl1 that we
have designated fibronectin-like domain (FLD). While, according to the initial hypothesis, proteins carrying the FLD tend
to adhere to the cell wall, our results argued against the idea of an involvement of the STRD in protein secretion that stemmed
from the presence of similar domains in different proteins secreted by yeast. The second objective of this work was to increase
the enzymatic repertoire by generating enzymes with new structural and functional properties. 相似文献
9.
Engineering <Emphasis Type="Italic">Escherichia coli</Emphasis> for efficient cellobiose utilization
Escherichia coli normally cannot utilize the β-glucoside sugar cellobiose as a carbon and energy source unless a stringent selection pressure
for survival is present. The cellobiose-utilization phenotype can be conferred by mutations in the two cryptic operons, chb and asc. In this study, the cellobiose-utilization phenotype was conferred to E. coli by replacing the cryptic promoters of these endogenous operons with a constitutive promoter. Evolutionary adaptation of the
engineered strain CP12CHBASC by repeated subculture in cellobiose-containing minimal medium led to an increase in the rate
of cellobiose uptake and cell growth on cellobiose. An efficient cellobiose-metabolizing E. coli strain would be of great importance over glucose-metabolizing E. coli for a simultaneous saccharification and fermentation process, as the cost of the process would be reduced by eliminating
one of the three enzymes needed to hydrolyze cellulose into simple sugars. 相似文献
10.
The 190-kDa Paenibacillus β-1,3-glucanase (LamA) contains a catalytic module of the glycoside hydrolase family 16 (GH16) and several auxiliary domains.
Of these, a discoidin domain (DS domain), present in both eukaryotic and prokaryotic proteins with a wide variety of functions,
exists at the carboxyl-terminus. To better understand the bacterial DS domain in terms of its structure and function, this
domain alone was expressed in Escherichia coli and characterized. The results indicate that the DS domain binds various polysaccharides and enhances the biological activity
of the GH16 module on composite substrates. We also investigated the importance of several conserved aromatic residues in
the domain's stability and substrate-binding affinity. Both were affected by mutations of these residues; however, the effect
on protein stability was more notable. In particular, the forces contributed by a sandwiched triad (W1688, R1756, and W1729)
were critical for the presumable β-sandwich fold. 相似文献
11.
Evolutionary Relationship of the Ligand-Gated Ion Channels and the Avermectin-Sensitive,Glutamate-Gated Chloride Channels 总被引:4,自引:0,他引:4
Demetrios K. Vassilatis Keith O. Elliston Philip S. Paress Michel Hamelin Joseph P. Arena James M. Schaeffer Lex H.T. Van der Ploeg Doris F. Cully 《Journal of molecular evolution》1997,44(5):501-508
Two cDNAs, GluClα and GluClβ, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin
class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707–711, 1994). Expression studies in Xenopus oocytes showed that GluClα and GluClβ have pharmacological profiles distinct from the glutamate-gated cation channels as
well as the γ-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of
related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined
the nucleotide sequence of the GluClα and GluClβ genes. In an attempt to understand the evolutionary relationship of these
channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic
analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several
intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position.
Phylogenetic analyses indicate that GluClα and GluClβ form a monophyletic subbranch in the ligand-gated ion channel superfamily
and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties
similar to GluClα and GluClβ, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride
channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination
with the distinct pharmacological properties demonstrate that GluClα and GluClβ belong to a discrete ligand-gated ion channel
family that may represent genes orthologous to the vertebrate glycine channels.
Received: 30 September 1996 / Accepted: 15 November 1996 相似文献
12.
Previous studies on the carbohydrate specificities of Erythrina cristagalli lectin (ECL) were mainly limited to analyzing the binding of oligo-antennary Galβ1→4GlcNAc (II). In this report, a wider range of recognition factors of ECL toward known mammalian ligands and glycans were examined by
enzyme-linked lectinosorbent and inhibition assays, using natural polyvalent glycotopes, and a glycan array assay. From the
results, it is shown that GalNAc was an active ligand, but its polyvalent structural units, in contrast to those of Gal, were
poor inhibitors. Among soluble natural glycans tested for 50% molecular mass inhibition, Streptococcus pneumoniae type 14 capsular polysaccharide of polyvalent II was the most potent inhibitor; it was 2.1 × 104, 3.9 × 103 and 2.4 × 103 more active than Gal, tri-antennary II and monomeric II, respectively. Most type II-containing glycoproteins were also potent inhibitors, indicating that special polyvalent II and Galβ1-related structures play critically important roles in lectin binding. Mapping all information available, it can
be concluded that: [a] Galβ1→4GlcNAc (II) and some Galβ1-related oligosaccharides, rather than GalNAc-related oligosaccharides, are the core structures for lectin
binding; [b] their polyvalent II forms within macromolecules are a potent recognition force for ECL, while II monomer and oligo-antennary II forms play only a limited role in binding; [c] the shape of the lectin binding domains may correspond to a cavity type with
Galβ1→4GlcNAc as the core binding site with additional one to four sugars subsites, and is most complementary to a linear
trisaccharide, Galβ1→4GlcNAcβ1→6Gal. These analyses should facilitate the understanding of the binding function of ECL.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
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Transducin (T) mediates vision in retinal rods by transmitting light signals detected by rhodopsin to a cGMP phosphodiesterase.
The flow of information relies on a subunit association/dissociation cycle of T regulated by a guanine nucleotide exchange/hydrolysis
reaction. 5′-[p-(Fluorosulfonyl)benzoyl] guanosine (FSBG) was synthesized and examined here as an affinity label for the guanine nucleotide
binding site of T. Although the relative binding affinity of FSBG to T was much lower than for GTP and β,γ-imido-guanosine
5′-triphosphate (GMPPNP), the incorporation of FSBG to T inhibited its light-dependent [3H] GMPPNP binding activity in a concentration dependent manner. Additionally, GDP, GTP and GTP analogs hindered the binding
of [3H] FSBG to T. These results demonstrated that FSBG could be used to specifically modify the active site of T. In addition,
FSBG was not capable of dissociating T from T:photoactivated rhodopsin complexes, suggesting that in this case FSBG is acting
as a GDP analog. 相似文献
15.
Corporeau C Groisillier A Jeudy A Barbeyron T Fleury E Fabioux C Czjzek M Huvet A 《Marine biotechnology (New York, N.Y.)》2011,13(5):971-980
The transforming growth factor (TGF)-β superfamily is a group of important growth factors involved in multiple processes such
as differentiation, cell proliferation, apoptosis and cellular growth. In the Pacific oyster Crassostrea gigas, the oyster gonadal (og) TGF-β gene was recently characterized through genome-wide expression profiling of oyster lines selected to be resistant or susceptible
to summer mortality. Og TGF-β appeared specifically expressed in the gonad to reach a maximum when gonads are fully mature, which singularly contrasts
with the pleiotropic roles commonly ascribed to most TGF-β family members. The function of og TGF-β protein in oysters is
unknown, and defining its role remains challenging. In this study, we develop a rapid bacterial production system to obtain
recombinant og TGF-β protein, and we demonstrate that og TGF-β is processed by furin to a mature form of the protein. This
mature form can be detected in vivo in the gonad. Functional inhibition of mature og TGF-β in the gonad was conducted by inactivation
of the protein using injection of antibodies. We show that inhibition of og TGF-β function tends to reduce gonadic area. We
conclude that mature og TGF-β probably functions as an activator of germ cells development in oyster. 相似文献
16.
The effect of mutations indnaK anddnaJ genes on the expression of two operons that are part of cysteine regulon was determined usingEscherichia coli strains harboringcysPTWA::lacZ andcysJIH::lacZ fusions. NulldnaJ, anddnaKdnaJ mutants were impaired in β-galactosidase expression from both fusions. Effecient complementation of this defect by wild-type
alleles present on a low-copy number plasmid was achieved. The presence of the pMH224 plasmid coding for CysB* protein defective in DNA binding lowered β-galactosidase expression fromcysPTWA::lacZ fusion strain harboring wild-typednaKdnaJ alleles but did not diminish enzyme expression in ΔdnaJ and ΔdnaKdnaJ strains. 相似文献
17.
To produce recombinant β-carotene in vitro, synthetic operons encoding genes governing its biosynthesis were engineered into Escherichia coli. Constructs harboring these operons were introduced into either a high-copy or low-copy cloning vector. β-Carotene production from these recombinant E. coli cells was either constitutive or inducible depending upon plasmid copy number. The most efficient β-carotene production was with the low-copy based vector. The process was increased incrementally from a 5 l to a 50 l fermentor and finally into a 300 l fermentor. The maximal β-carotene yields achieved using the 50 l and 300 l fermentor were 390 mg l−1 and 240 mg l−1, respectively, with overall productivities of 7.8 mg l−1 h−1 and 4.8 mg l−1 h−1. 相似文献
18.
Walid Saibi Salma Abdeljalil Ali Gargouri 《World journal of microbiology & biotechnology》2011,27(8):1765-1774
Stachybotrys microspora is a filamentous fungus characterized by the secretion of multiple β-glucosidases. The production of these enzymes was studied
under culture with variable carbon sources. The highest activity was produced on glucose (0.66 U ml−1) whereas galactose, lactose, cellobiose, Avicel cellulose, carboxymethylcellulose (CMC), wheat bran and gruel allowed intermediate
production levels ranging between 0.08 and 0.48 U ml−1. The zymogram analysis showed that complex sugars such as Avicel cellulose and CMC induced the expression of several β-glucosidases
whereas all tested simple sugars (mono and disaccharides) induced the expression of one single β-glucosidase, each time different.
The most efficient β-glucosidase named bglG was produced on glucose which continues to be, at the same time, its strong inhibitor.
The bglG N-terminal sequence confirmed that it is a novel β-glucosidase. According to its large molecular weight, this enzyme
was assumed to belong to family 3 of β-glucosidases. RT-PCR analysis showed that family 3 expressions were induced on glucose
while those of family 1 were repressed. Finally, with the use of different combinations of glucose and various carbon sources
at different ratio, we showed that such sources direct the differential expression of β-glucosidases in S. microspora since our strain co-produced the β-glucosidases corresponding to each carbon source. 相似文献
19.
Leonid S. Brown 《BBA》2014
Since the discovery of proteorhodopsins, the ubiquitous marine light-driven proton pumps of eubacteria, a large number of other eubacterial rhodopsins with diverse structures and functions have been characterized. Here, we review the body of knowledge accumulated on the four major groups of eubacterial rhodopsins, with the focus on their biophysical characterization. We discuss advances and controversies on the unique eubacterial sensory rhodopsins (as represented by Anabaena sensory rhodopsin), proton-pumping proteorhodopsins and xanthorhodopsins, as well as novel non-proton ion pumps. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks. 相似文献
20.
Ningzhi Xu Omar Coso Daruka Mahadevan Antonio De Blasi Paul K. Goldsmith William F. Simonds J. Silvio Gutkind 《Cellular and molecular neurobiology》1996,16(1):51-59
Summary 1. The noncatalytic domain of Ras-GAP can affect signaling through G protein-coupled receptors by a poorly understood mechanism.
2. In this study, fusion proteins containing elements of the noncatalytic domain ofras-GAP were examined for their ability to bindβγ subunits of heterotrimeric G proteins and phosphotyrosine-containing polypeptides.
3. Our results demonstrate that purifiedβγ dimers associated with bacterially expressed GAP proteins and that this association does not require SH2 or SH3 domains but
is dependent on the presence of the GAP pleckstrin-homology (PH) domain. In contrast, only the SH2 domains are necessary for
binding to tyrosine phosphorylated proteins.
4. These findings raise the possibility that heterotrimeric G proteins might affect functioning ofras-like proteins throughβγ subunits acting on their regulatory molecules. 相似文献