共查询到20条相似文献,搜索用时 46 毫秒
1.
Murakami K Yokoyama S Murata N Ozawa Y Irie K Shirasawa T Shimizu T 《Biochemical and biophysical research communications》2011,410(1):34-39
tRNA-guanine transglycosylases (TGTs) are responsible for incorporating 7-deazaguanine-modified bases into certain tRNAs in eubacteria (preQ1), eukarya (queuine) and archaea (preQ0). In each kingdom, the specific modified base is different. We have found that the eubacterial and eukaryal TGTs have evolved to be quite specific for their cognate heterocyclic base and that Cys145 (Escherichia coli) is important in recognizing the amino methyl side chain of preQ1 (Chen et al., Nuc. Acids Res. 39 (2011) 2834 [15]). A series of mutants of the E. coli TGT have been constructed to probe the role of three other active site amino acids in the differential recognition of heterocyclic substrates. These mutants have also been used to probe the differential inhibition of E. coli versus human TGTs by pteridines. The results indicate that mutation of these active site amino acids can “open up” the active site, allowing for the binding of competitive pteridine inhibitors. However, even the “best” of these mutants still does not recognize queuine at concentrations up to 50 μM, suggesting that other changes are necessary to adapt the eubacterial TGT to incorporate queuine into RNA. The pteridine inhibition results are consistent with an earlier hypothesis that pteridines may regulate eukaryal TGT activity (Jacobson et al., Nuc. Acids Res. 9 (1981) 2351 [8]). 相似文献
2.
N.M. Goodey M.T. Alapa D.F. Hagmann S.G. Korunow A.K. Mauro K.S. Kwon S.M. Hall 《Biochemical and biophysical research communications》2011,(3):442
A fluorescently-labeled, conformationally-sensitive Bacillus stearothermophilus (Bs) dihydrofolate reductase (DHFR) (C73A/S131CMDCC DHFR) was developed and used to investigate kinetics and protein conformational motions associated with methotrexate (MTX) binding. This construct bears a covalently-attached fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) attached at a distal cysteine, introduced by mutagenesis. The probe is sensitive to the local molecular environment, reporting on changes in the protein structure associated with ligand binding. Intrinsic tryptophan fluorescence of the unlabeled Bs DHFR construct (C73A/S131C DHFR) also showed changes upon MTX association. Stopped-flow analysis of all data can be understood by invoking the presence of two native state DHFR conformers that bind to MTX at different rates (20.2 and 0.067 μM−1 s−1), similar to previously published findings for Escherichia coli DHFR. Probe fluorescence of C73A/S131CMDCC DHFR predominantly reports on MTX binding to one of the conformers while intrinsic tryptophan fluorescence of C73A/S131C DHFR reports on binding to the other conformer. This study demonstrates the use of an extrinsic fluorophore attached to a distal region to investigate ligand binding interactions that are not experimentally accessible via intrinsic tryptophan fluorescence alone. The thermostability of C73A/S131CMDCC DHFR provides an important new tool with applications for investigating the temperature dependence of DHFR conformational changes associated with binding and catalysis. 相似文献
3.
This study describes an analysis of different treatments that influence the relative content and the midpoint potential of HP Cyt b559 in PS II membrane fragments from higher plants. Two basically different types of irreversible modification effects are distinguished: the HP form of Cyt b559 is either predominantly affected when the heme group is oxidized (“O-type” effects) or when it is reduced (“R-type” effects). Transformation of HP Cyt b559 to lower potential redox forms (IP and LP forms) by the “O-type” mechanism is induced by high pH and detergent treatments. In this case the effects consist of a gradual decrease in the relative content of HP Cyt b559 while its midpoint potential remains unaffected. Transformation of HP Cyt b559 via an “R-type” mechanism is caused by a number of exogenous compounds denoted L: herbicides, ADRY reagents and tetraphenylboron. These compounds are postulated to bind to the PS II complex at a quinone binding site designated as QC which interacts with Cyt b559 and is clearly not the QB site. Binding of compounds L to the QC site when HP Cyt b559 is oxidized gives rise to a gradual decrease in the Em of HP Cyt b559 with increasing concentration of L (up to 10 Kox(L) values) while the relative content of HP Cyt b559 is unaffected. Higher concentrations of compounds L required for their binding to QC site when HP Cyt b559 is reduced (described by Kred(L)) induce a conversion of HP Cyt b559 to lower potential redox forms (“R-type” transformation). Two reaction pathways for transitions of Cyt b559 between the different protein conformations that are responsible for the HP and IP/LP redox forms are proposed and new insights into the functional regulation of Cyt b559 via the QC site are discussed. 相似文献
4.
Fluorescent proteins homologous to green fluorescent protein (avGFP) display pronounced spectral variability due to different chromophore structures and variable chromophore interactions with the surrounding amino acids. To gain insight into the structural basis for yellow emission, the 3D structure of phiYFP (λem = 537 nm), a protein from the sea medusa Phialidium sp., was built by a combined homology modeling – mass spectrometry approach. Mass spectrometry of the isolated chromophore-bearing peptide reveals that the chromophore of phiYFP is chemically identical to that of avGFP (λem = 508 nm). The experimentally acquired chromophore structure was combined with the homology-based model of phiYFP, and the proposed 3D structure was used as a starting point for identification of the structural features responsible for yellow fluorescence. Mutagenesis of residues in the local chromophore environment of phiYFP suggests that multiple factors cooperate to establish the longest-wavelength emission maximum among fluorescent proteins with an unmodified GFP-like chromophore. 相似文献
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6.
A study of an enzyme that reacts with the sulfenic acid produced by the alliinase in Petiveria alliacea L. (Phytolaccaceae) to yield the P. alliacea lachrymator (phenylmethanethial S-oxide) showed the protein to be a dehydrogenase. It functions by abstracting hydride from sulfenic acids of appropriate structure to form their corresponding sulfines. Successful hydride abstraction is dependent upon the presence of a benzyl group on the sulfur to stabilize the intermediate formed on abstraction of hydride. This dehydrogenase activity contrasts with that of the lachrymatory factor synthase (LFS) found in onion, which catalyzes the rearrangement of 1-propenesulfenic acid to (Z)-propanethial S-oxide, the onion lachrymator. Based on the type of reaction it catalyzes, the onion LFS should be classified as an isomerase and would be called a “sulfenic acid isomerase”, whereas the P. alliacea LFS would be termed a “sulfenic acid dehydrogenase”. 相似文献
7.
The thiol oxidase Erv1 and the redox-regulated receptor Mia40/Tim40 are components of a disulfide relay system which mediates import of proteins into the intermembrane space (IMS) of mitochondria. Here we report that Erv1 requires Mia40 for its import into mitochondria. After passage across the translocase of the mitochondrial outer membrane Erv1 interacts via disulfide bonds with Mia40. Erv1 does not contain twin “CX3C” or twin “CX9C” motifs which are crucial for import of typical substrates of this pathway and it does not need two “CX2C” motifs for import into mitochondria. Thus, Erv1 represents an unusual type of substrate of the Mia40-dependent import pathway. 相似文献
8.
Fabrini R Bocedi A Dawood KF Turella P Stella L Parker MW Pedersen JZ Federici G Antonini G Ricci G 《FEBS letters》2011,585(2):341-345
Glutathione transferase reaches 0.5–0.8 mM concentration in the cell so it works in vivo under the unusual conditions of, [S] ? [E]. As glutathione transferase lowers the pKa of glutathione (GSH) bound to the active site, it increases the cytosolic concentration of deprotonated GSH about five times and speeds its conjugation with toxic compounds that are non-typical substrates of this enzyme. This acceleration becomes more efficient in case of GSH depletion and/or cell acidification. Interestingly, the enzymatic conjugation of GSH to these toxic compounds does not require the assumption of a substrate–enzyme complex; it can be explained by a simple bimolecular collision between enzyme and substrate. Even with typical substrates, the astonishing concentration of glutathione transferase present in hepatocytes, causes an unusual “inverted” kinetics whereby the classical trends of v versus E and v versus S are reversed. 相似文献
9.
The state transition in cyanobacteria is a long-discussed topic of how the photosynthetic machine regulates the excitation energy distribution in balance between the two photosystems. In the current work, whether the state transition is realized by “mobile phycobilisome (PBS)” or “energy spillover” has been clearly answered by monitoring the spectral responses of the intact cells of the cyanobacterium Spirulina platensis. Firstly, light-induced state transition depends completely on a movement of PBSs toward PSI or PSII while the redox-induced one on not only the “mobile PBS” but also an “energy spillover”. Secondly, the “energy spillover” is triggered by dissociation of PSI trimers into the monomers which specially occurs under a case from light to dark, while the PSI monomers will re-aggregate into the trimers under a case from dark to light, i.e., the PSI oligomerization is reversibly regulated by light switch on and off. Thirdly, PSI oligomerization is regulated by the local H+ concentration on the cytosol side of the thylakoid membranes, which in turn is regulated by light switch on and off. Fourthly, PSI oligomerization change is the only mechanism for the “energy spillover”. Thus, it can be concluded that the “mobile PBS” is a common rule for light-induced state transition while the “energy spillover” is only a special case when dark condition is involved. 相似文献
10.
Ey, P. L., and Darby, J. M. 1998.Giardia intestinalis: Conservation of the variant-specific surface protein VSP417-1 (TSA417) and identification of a divergent homologue encoded at a duplicated locus in genetic Group II isolates.Experimental Parasitology90, 250–261. The stability of the gene encoding TSA417, a 72-kDa variant-specific surface protein (VSP) produced by trophozoites ofGiardia intestinalisisolate WB-C6, was investigated in isolates of similar (Assemblage A / Group I) or distinct (Assemblage A / Group II) genotype. Using primers specific for the WB-C6tsa417gene, DNA amplified in polymerase chain reactions from genomic DNA indicated the presence, in every isolate, of an intact coding sequence possessing conserved restriction sites diagnostic for this locus (herein designatedvsp417-1). Sequence analysis of the DNA amplified from the genomes of genetic Group I (“A-I”) isolates revealed complete identity with the published WB-C6tsa417(vsp417-1A-I) sequence. Equivalent products, amplified from the genomes of genetic Group II (“A-II”) isolates, similarly yielded an invariant and apparently allelic 2142-bp coding sequence (designatedvsp417-1A-II) possessing 79% nucleotide identity withvsp417-1A-Iand polymorphisms unique to Group II organisms. The encoded polypeptides (VSP417-1A-Iand VSP417-1A-II) are identical at 75% of amino acid positions. Substitutions are concentrated within the N-terminal portions of the proteins, but the overall structure of VSP417-1 has changed little during the evolution of the Group I and Group II genotypes from their common clonal ancestor. An additional 0.7-kb DNA, representing a separate locus (vsp417-5) encoding a 22.3-kDa VSP, was amplified from genetic Group II genomes exclusively but only using particular primer combinations. Thevsp417-5A-IIgene exhibits >85% sequence identity with the 5′ and 3′ segments ofvsp417-1A-Iandvsp417-1A-IIbut it lacks a 1482-bp segment that comprises the central portion of thevsp417-1 locus. Excision of this segment seems to have occurred by intragenic recombination, possibly initiated by a stem loop formed between palindromic sequences which border the 1482-bp segment withinvsp417-1 but which are contiguous invsp417-5A-II. The detection by Southern hybridization of additional genomic sequences that share homology with these genes reveals the existence in these two genotypes of a distinctive “vsp417” gene subset. 相似文献
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12.
Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed Km values of 41 μM and 34 μM for adenosine and 2′-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with Ki value of 0.5 μM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional –SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA. 相似文献
13.
Barbara Demmig-Adams William W. Adams III Klaus Winter Angelika Meyer Ulrich Schreiber João S. Pereira Almuth Krüger Franz-Christian Czygan Otto L. Lange 《Planta》1989,177(3):377-387
During the midday depression of net CO2 exchange in the mediterranean sclerophyllous shrub Arbutus unedo, examined in the field in Portugal during August of 1987, several parameters indicative of photosynthetic competence were strongly and reversibly affected. These were the photochemical efficiency of photosystem (PS) II, measured as the ratio of variable to maximum chlorophyll fluorescence, as well as the photon yield and the capacity of photosynthetic O2 evolution at 10% CO2, of which the apparent photon yield of O2 evolution was most depressed. Furthermore, there was a strong and reversible increase in the content of the carotenoid zeaxanthin in the leaves that occurred at the expense of both violaxanthin and -carotene. Diurnal changes in fluorescence characteristics were interpreted to indicate three concurrent effects on the photochemical system. First, an increase in the rate of radiationless energy dissipation in the antenna chlorophyll, reflected by changes in 77K fluorescence of PSII and PSI as well as in chlorophyll a fluorescence at ambient temperature. Second, a state shift characterized by an increase in the proportion of energy distributed to PSI as reflected by changes in PSI fluorescence. Third, an effect lowering the photon yield of O2 evolution and PSII fluorescence at ambient temperature without affecting PSII fluorescence at 77K which would be expected from a decrease in the activity of the water splitting enzyme system, i.e. a donor side limitation.Abbreviations and symbols ci
concentration of CO2 within the leaf
- Fo
instantaneous fluorescence emission
- FM
maximum fluorescence emission
- Fv
variable fluorescence emission
- PFD
photon flux density (400–700 nm)
- PSI, II
photosystem I, II
- TL
leaf temperature 相似文献
14.
J.Michael Elliott Mark Ultsch Joshua Lee Raymond Tong Kentaro Takeda Christoph Spiess Charles Eigenbrot Justin M. Scheer 《Journal of molecular biology》2014
Bispecific antibody and antibody-like molecules are of wide interest as potential therapeutics that can recognize two distinct targets. Among the variety of ways such molecules have been engineered is by creating “knob” and “hole” heterodimerization sites in the CH3 domains of two antibody heavy chains. The molecules produced in this manner maintain their biological activities while differing very little from the native human IgG sequence. To better understand the knob-into-hole interface, the molecular mechanism of heterodimerization, and to engineer Fc domains that could improve the assembly and purity of heterodimeric reaction products, we sought crystal structures of aglycosylated heterodimeric and homodimeric “knob” and “hole” Fc fragments derived from bacterial expression. The structure of the knob-into-hole Fc was determined at 2.64 Å. Except for the sites of mutation, the structure is very similar to that of the native human IgG1 Fc, consistent with a heterodimer interaction kinetic KD of < 1 nM. Homodimers of the “knob” and “hole” mutants were also obtained, and their X-ray structures were determined at resolutions 2.5 Å and 2.1 Å, respectively. Both kinds of homodimers adopt a head-to-tail quaternary structure and thus do not contain direct knob/knob or hole/hole CH3 interactions. The head-to-tail arrangement was disfavored by adding site-directed mutations at F241 and F243 in the CH2 domains, leading to increases in both rate and efficiency of bispecific (heterodimer) assembly. 相似文献
15.
Nicolas Puff Chiho Watanabe Michel Seigneuret Miglena I. Angelova Galya Staneva 《生物化学与生物物理学报:生物膜》2014
Lipid rafts are assumed to undergo biologically important size-modulations from nanorafts to microrafts. Due to the complexity of cellular membranes, model systems become important tools, especially for the investigation of the factors affecting “raft-like” Lo domain size and the search for Lo nanodomains as precursors in Lo microdomain formation. Because lipid compositional change is the primary mechanism by which a cell can alter membrane phase behavior, we studied the effect of the ganglioside GM1 concentration on the Lo/Ld lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1 mol % abolishes the formation of the micrometer-scale Lo domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a Lo/Ld lateral phase separation taking place below the optical resolution. This nanoscale phase separation is revealed by fluorescence spectroscopy, including C12NBD-PC self-quenching and Laurdan GP measurements, and is supported by Gaussian spectral decomposition analysis. The temperature of formation of nanoscale Lo phase domains over an Ld phase is determined, and is shifted to higher values when the GM1 content increases. A “morphological” phase diagram could be made, and it displays three regions corresponding respectively to Lo/Ld micrometric phase separation, Lo/Ld nanometric phase separation, and a homogeneous Ld phase. We therefore show that a lipid only-based mechanism is able to control the existence and the sizes of phase-separated membrane domains. GM1 could act on the line tension, “arresting” domain growth and thereby stabilizing Lo nanodomains. 相似文献
16.
María Carmen Pallarés Carlos Marcuello Laura Botello-Morte Andrés González María Francisca Fillat Anabel Lostao 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(3):623-631
Fur (ferric uptake regulator) proteins are involved in the control of a variety of processes in most prokaryotes. Although it is assumed that this regulator binds its DNA targets as a dimer, the way in which this interaction occurs remains unknown. We have focused on FurA from the cyanobacterium Anabaena sp. PCC 7120. To assess the molecular mechanism by which FurA specifically binds to “iron boxes” in PfurA, we examined the topology arrangement of FurA–DNA complexes by atomic force microscopy. Interestingly, FurA–PfurA complexes exhibit several populations, in which one is the predominant and depends clearly on the regulator/promoter ratio on the environment. Those results together with EMSA and other techniques suggest that FurA binds PfurA using a sequential mechanism: (i) a monomer specifically binds to an “iron box” and bends PfurA; (ii) two situations may occur, that a second FurA monomer covers the free “iron box" or that joins to the previously used forming a dimer which would maintain the DNA kinked; (iii) trimerization in which the DNA is unbent; and (iv) finally undergoes a tetramerization; the next coming molecules cover the DNA strands unspecifically. In summary, the bending appears when an “iron box” is bound to one or two molecules and decreases when both “iron boxes” are covered. These results suggest that DNA bending contributes at the first steps of FurA repression promoting the recruitment of new molecules resulting in a fine regulation in the Fur-dependent cluster associated genes. 相似文献
17.
Somnath Mukherjee 《Journal of molecular biology》2010,401(5):949-968
The dreaded pathogen Staphylococcus aureus is one of the causes of morbidity and mortality worldwide. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), one of the key glycolytic enzymes, is irreversibly oxidized under oxidative stress and is responsible for sustenance of the pathogen inside the host. With an aim to elucidate the catalytic mechanism and identification of intermediates involved, we describe in this study different crystal structures of GAPDH1 from methicillin-resistant S. aureus MRSA252 (SaGAPDH1) in apo and holo forms of wild type, thioacyl intermediate, and ternary complexes of active-site mutants with physiological substrate d-glyceraldehyde-3-phosphate (G3P) and coenzyme NAD+. A new phosphate recognition site, “new Pi” site, similar to that observed in GAPDH from Thermotoga maritima, is reported here, which is 3.40 Å away from the “classical Pi” site. Ternary complexes discussed are representatives of noncovalent Michaelis complexes in the ground state. d-G3P is bound to all the four subunits of C151S.NAD and C151G.NAD in more reactive hydrate (gem-di-ol) form. However, in C151S + H178N.NAD, the substrate is bound to two chains in aldehyde form and in gem-di-ol form to the other two. This work reports binding of d-G3P to the C151G mutant in an inverted manner for the very first time. The structure of the thiaocyl complex presented here is formed after the hydride transfer. The C3 phosphate of d-G3P is positioned at the “Ps” site in the ternary complexes but at the “new Pi” site in the thioacyl complex and C1-O1 bond points opposite to His178 disrupting the alignment between itself and NE2 of His178. A new conformation (Conformation I) of the 209-215 loop has also been identified, where the interaction between phosphate ion at the “new Pi” site and conserved Gly212 is lost. Altogether, inferences drawn from the kinetic analyses and crystal structures suggest the “flip-flop” model proposed for the enzyme mechanism. 相似文献
18.
In this paper, we show the unique role of bicarbonate ion in stimulating the electron transfer of photosystem II (PS II) in formate-treated leaf discs from spinach. This is referred to as the bicarbonate effect and is independent of the role of CO2 in CO2 fixation. It is shown to have two sites of action:
The first site of inhibition by formate-treatment is detected by the decrease of the rate of oxygen evolution and the simultaneous quenching of the variable chlorophyll a (Chl a) fluorescence of leaf discs infiltrated with 100 mM formate for about 10 s followed by storage for 10 min in dark. This is referred to as short-term formate treatment. Addition of bicarbonate reverses this short-term formate effect and restores fully both Chl a fluorescence and oxygen evolution rate. Reversible quenching of variable Chl a fluorescence of heated and short-term formate treated leaf discs, in the presence of hydroxylamine as an artificial electron donor to PS II, is also observed. This suggests that the first site of action of the anion effect is indeed between the site of donation of hydroxylamine to PS II (i.e. Z or D) and QA. The second site of the effect, where bicarbonate depletion has its most dramatic effect, as well known in thylakoids, is shown by an increase of Chl a fluorescence of leaf discs infiltrated with 100 mM formate for about 10 min followed by storage for 10 min in dark. This is referred to as the long-term formate treatment. Addition of bicarbonate fully restores the variable Chl a fluorescence of these leaf discs. Chl a fluorescence transient of DCMU-infiltrated (10 min) leaf discs is similar to that of long-term formate-treated one suggesting that the absence of bicarbonate, like the presence of DCMU, inhibits the electron flow beyond QA. 相似文献
(1) | the first, described here for the first time, stimulates the electron flow between the hydroxylamine donation site (Z or D) and QA, the first plastoquinone electron acceptor and |
(2) | the other accelerates the electron flow beyond QA, perhaps at the QA QB complex, where QB is the second plastoquinone electron acceptor. |
19.
Arvanitoyannis IS Bouletis AD Papa EA Gkagtzis DC Hadjichristodoulou C Papaloucas C 《Anaerobe》2011,17(6):303-306
Fresh rocket “Eruca Sativa” and lettuce “Lollo Verde” leaves were stored with the addition of olive oil and wine vinegar “Aceto balsamico di Modena” under modified atmosphere packaging (MAP) (5% O2/10% CO2/85% N2 for MAP A and 2% O2/5% CO2/93% N2 for MAP B). The microbial (mesophilic, psychrotrophic bacteria and Enterobacteriacae), physical (color and firmness) and sensory parameters of samples were studied in relation to storage time (up to 10 days at 5 ± 1 °C). The effect of wine vinegar and the application of both MAP treatments reduced the growth of all bacteria populations (p < 0.05). Samples with olive oil stored under MAP A gave the best score for overall impression (3 and 2.1 for MAP A and B respectively at the 9th day of storage) while the addition of vinegar limited sensory shelf-life to 3 days (p < 0.05). Firmness was negatively affected by wine vinegar while samples with olive oil stored under MAP A maintained firmness close to normal. Color attributes were maintained better under both MAP treatments (p < 0.05). 相似文献
20.
Mycobacterium tuberculosis (Mtb) has a highly complex cell wall, which is required for both bacterial survival and infection. Cell wall biosynthesis is dependent on decaprenyl diphosphate as a glyco-carrier, which is hence an essential metabolite in this pathogen. Previous biochemical studies indicated (E)-geranyl diphosphate (GPP) is required for the synthesis of decaprenyl diphosphate. Here we demonstrate that Rv0989c encodes the “missing” GPP synthase, representing the first such enzyme to be characterized from bacteria, and which presumably is involved in decaprenyl diphosphate biosynthesis in Mtb. Our investigation also has revealed previously unrecognized substrate plasticity of the farnesyl diphosphate synthases from Mtb, resolving previous discrepancies between biochemical and genetic studies of cell wall biosynthesis. 相似文献