共查询到20条相似文献,搜索用时 15 毫秒
1.
A. A. Levy M. Fridlender U. Hanania E. Rubin Y. Sitrit 《Molecular & general genetics : MGG》1996,251(4):436-441
Specific binding ofNicotiana nuclear protein(s) to subterminal regions of theAc transposable element was detected using gel mobility shift assays. A sequence motif (GGTAAA) repeated in both terminal regions ofAc, was identified as the protein binding site. Mutation of two nucleotides in this motif was sufficient to abolish binding. Based on a series of competition assays, it is deduced that there is cooperative binding between two repeats, each similar to the GGTAAA motif. The binding protein is probably similar to a previously characterized maize protein which binds to a GGTAAA-containing motif located in the ends ofMutator. Moreover, we show that DNA fromDs1 competes for protein binding toAc termini, and we show, by sequence analysis, that GGTAAA binding sites are present in the terminal region ofTgm1, Tpn1, En/Spm, Tam3 andDs1-like elements. This suggests that the binding protein(s) might be involved in the transposition process. 相似文献
2.
C. J. Lenz J. W. Dillwith G. M. Chippendale 《Archives of insect biochemistry and physiology》1986,3(1):61-73
The properties of the high-affinity low molecular weight juvenile hormone (JH) binding protein present in the hemolymph of larvae of five species of pyralid moths, a noctuid moth, and a sphingid moth were compared. The pyralid moths exhibit a facultative diapause as last-instar larvae. The species employed were the southwestern corn borer, Diatraea grandiosella, the southern cornstalk borer, Diatraea crambidoides, the sugarcane borer, Diatraea saccharalis, the European corn borer, Ostrinia nubilalis, the sunflower moth, Homoeosoma electellum, the cabbage looper, Trichoplusia ni, and the tobacco hornworm, Manduca sexta. The binding characteristics of the proteins were determined using saturation binding assays and competitive binding assays. The dissociation constants of JH I, JH II, and JH III for the binding protein of all the species varied from 0.8 x 10?7 M to 2.8 x 10?7 M. Calibrated gel filtration showed that the binding protein of all the species had apparent molecular weights ranging from 29,000 to 31,000. Electrophoresis in 7% acrylamide gels revealed that the relative mobilities of the binding proteins ranged from 0.33 to 0.43. Isoelectric focusing showed that the binding proteins had isoelectric points between 4.4 and 5.0. 相似文献
3.
Studies on the possible role of protein phosphorylation in the transduction of the ethylene signal 总被引:4,自引:0,他引:4
A. W. Berry D. S. C. Cowan N. V. J. Harpham R. J. Hemsley G. V. Novikova A. R. Smith M. A. Hall 《Plant Growth Regulation》1996,18(1-2):135-141
Previous work in our laboratory has demonstrated the existence of high affinity binding sites for the plant growth regulator ethylene. The ethylene binding protein (EBP), from Phaseolus cotyledons, shows many of the characteristics of a functional receptor for ethylene, has been purified on SDS-PAGE and polyclonal antibodies raised in rabbits. Current work involves the investigation of the ethylene transduction signal in a number of ethylene-responsive tissues.In peas, it has been shown that ethylene promotes the phosphorylation of specific proteins of similar molecular weight to the EBP from Phaseolus. Such ethylene-induced phosphorylation can be inhibited by the ethylene antagonist, 2,5-NBD. The antibodies raised to the EBP from Phaseolus have been shown to immunoprecipitate 32P-labelled proteins from membrane protein preparations obtained from pea tissue. Studies on ethylene binding in pea have also shown that the binding of ethylene may be regulated by phosphorylation. Thus, under conditions which promote phosphorylation, binding is inhibited, whereas the reverse is true under conditions which enhance dephosphorylation.Further work is described which examines the effect of protein kinase, protein phosphatase and calcium channel inhibitors on ethylene-induced phosphorylation in peas, together with wild-type (WT) and ethylene insensitive (eti) mutants of Arabidopsis thaliana. The effects of these treatments can be monitored in vivo using the ethylene-induced triple response as a screen. Furthermore, the protein profiles of such treated seedlings can then be compared by labelling protein extracts with 32P and subjecting the samples to SDS-PAGE followed by autoradiography. 相似文献
4.
Ruth Levitz Ilan Friedberg Ruth Brucker Asora Fux Ezra Yagil 《Molecular & general genetics : MGG》1985,200(1):118-122
Summary The periplasmic phosphate binding protein is a product of the phoS gene and is an essential component of the phosphate specific transport (PST) system, which mediates Pi uptake in Escherichia coli. The binding of Pi to periplasmic protein(s) and the kinetic parameters of Pi uptake were studied in phoT and pstB mutants of E. coli. These mutants are impaired in Pi uptake but have a periplasmic Pi-binding protein whose Pi-binding acpacity was estimated by the retention kinetics. The Pi-binding activity in two pstB mutants was found to be weaker as compared to phoT9 and the wild type. The K
D values for Pi binding to periplasmic protein were determined by equilibrium dialysis. In the pstB mutants the K
D value was found to be 9–31 times higher than the values obtained for the wild type and the phoT mutant. The apparent K
m values for Pi uptake in one pstB mutant is 14.3 times higher than in the wild type. V
max of the mutant is 8.3 times lower that of the wild type. The data indicate that pstB, an essential gene of the PST transport system, is promoting the binding capacity of the Pi-binding protein.Abbreviations AP
alkaline phosphatase
- Pi
inorganic orthophosphate
- Km
kanamycin 相似文献
5.
L. Laurenzio D. G. Scraba W. Paranchych L. S. Frost 《Molecular & general genetics : MGG》1995,247(6):726-734
The origin of transfer (oriT) of the IncFV plasmid pED208 contains a region with three binding sites for both the plasmid-encoded TraM protein and the integration host factor (IHF) of Escherichia coli, a sequence-specific DNA-binding protein. One region, containing overlapping TraM and IHF binding sites, could be interpreted as containing two binding sites for each protein. Using gel retardation assays, an affinity constant for IHF binding to the three main sites was estimated in the presence and absence of 0.1 M potassium glutamate, which increased the avidity of IHF binding to the weaker sites by two orders of magnitude. DNase I protection analyses and electron microscopy were used to determine the affinity of IHF for oriT-containing DNA in the presence and absence of TraM. The binding of IHF and TraM was found to be non-cooperative by the two techniques employed. Electron microscopy also demonstrated that IHF bent the oriT region in a manner consistent with its previously determined mode of action, while TraM had no discernible effect on the appearance of the DNA. This suggested that IHF and TraM interact with a 295 by sequence in the oriT region and organize it into a higher order structure that may have a role in the initiation of DNA transfer and control of traM expression. 相似文献
6.
The octamer binding site, which is located immediately upstream of the poorly conserved DRA TATA sequence, is important fof high levels of expression of this human major histocompatibility class II gene in B cells. In this study, we demonstrate that the substitution of the DRA TATA sequence with the TATA box from the adenovirus E1b promoter revomes the requirements for the octamer binding site for high levels of expression from DRA promoter. Since only the TATA box from the E1b but bot the DRA promoters binds the TATA binding protein, we conclude that the octamer binding site helps to recruit TBP to the DRA promoter. 相似文献
7.
Synthetic peptides mimicking the binding site of human acetylcholinesterase for its inhibitor fasciculin 2
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Uwe Kafurke Ariel Erijman Yonatan Aizner Julia M. Shifman Jutta Eichler 《Journal of peptide science》2015,21(9):723-730
Molecules capable of mimicking protein binding and/or functional sites present useful tools for a range of biomedical applications, including the inhibition of protein–ligand interactions. Such mimics of protein binding sites can currently be generated through structure‐based design and chemical synthesis. Computational protein design could be further used to optimize protein binding site mimetics through rationally designed mutations that improve intermolecular interactions or peptide stability. Here, as a model for the study, we chose an interaction between human acetylcholinesterase (hAChE) and its inhibitor fasciculin‐2 (Fas) because the structure and function of this complex is well understood. Structure‐based design of mimics of the hAChE binding site for Fas yielded a peptide that binds to Fas at micromolar concentrations. Replacement of hAChE residues known to be essential for its interaction with Fas with alanine, in this peptide, resulted in almost complete loss of binding to Fas. Computational optimization of the hAChE mimetic peptide yielded a variant with slightly improved affinity to Fas, indicating that more rounds of computational optimization will be required to obtain peptide variants with greatly improved affinity for Fas. CD spectra in the absence and presence of Fas point to conformational changes in the peptide upon binding to Fas. Furthermore, binding of the optimized hAChE mimetic peptide to Fas could be inhibited by hAChE, providing evidence for a hAChE‐specific peptide–Fas interaction. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
8.
Summary Nuclear protein extracts from Mu-active, Mu-inactive and non-Mutator lines of maize were used to identify the binding sites for maize nuclear proteins in the terminal inverted repeats (TIR) of the Mul transposable element. We found binding activities of nuclear proteins that specifically interact with both TIRs of the Mu1 element. DNase I footprinting was performed to localize the binding sites. We found that the nuclear proteins from Mu-active lines and non-Mu lines bound to the Mu1 TIR at two different sites, i.e. a 13 by sequence (CGGGAACGGTAAA, designated as site I) and another 8 by sequence (CGGCGTCT, designated as site II). However, the nuclear proteins from Mu-inactive lines bound only one of these sites, i.e. site I. Mobility shift assays with synthetic oligonucleotides containing site I and 11 respectively confirmed the specificities of these binding activities. Site I was shown to be an imperfect direct repeat of a hexamer binding site (CGGGAA CGGTAA). Oligonucleotides containing either of the hexamers showed specific binding activity to nuclear protein from both Mu-active and Mu-inactive lines. The possible role of these proteins in Mu transposition is discussed. 相似文献
9.
10.
Flagella are nanofibers that drive bacterial movement. The filaments are generally composed of thousands of tightly packed flagellin subunits with a terminal cap protein, named FliD. Here, we report that the FliD protein of the bacterial pathogen Campylobacter jejuni binds to host cells. Live‐cell imaging and confocal microscopy showed initial contact of the bacteria with epithelial cells via the flagella tip. Recombinant FliD protein bound to the surface of intestinal epithelial cells in a dose‐dependent fashion. Search for the FliD binding site on the host cell using cells with defined glycosylation defects indicated glycosaminoglycans as a putative target. Heparinase treatment of wild type cells and an excess of soluble heparin abolished FliD binding. Binding assays showed direct and specific binding of FliD to heparin. Addition of an excess of purified FliD or heparin reduced the attachment of viable C. jejuni to the host cells. The host cell binding domain of FliD was mapped to the central region of the protein. Overall, our results indicate that the C. jejuni flagellar tip protein FliD acts as an attachment factor that interacts with cell surface heparan sulfate glycosaminoglycan receptors. 相似文献
11.
Protein F: an adhesin of Streptococcus pyogenes binds fibronectin via two distinct domains 总被引:18,自引:4,他引:14
Shiomo Sela Adi Aviv Aviva Tovi Israel Burstein Michael G. Caparon Emanuel Hanski 《Molecular microbiology》1993,10(5):1049-1055
The binding of Streptococcus pyogenes to fibronectin (FN) enables the adherence of this pathogen to target epithelial cells, which is the first necessary step for initiation of infection. Binding is mediated by a bacterial surface protein termed protein F. Here we provide the complete structure of protein F and identify two domains responsible for binding to fibronectin. The first domain is located towards the C-terminal end of the molecule and is composed of five repeats of 37 amino acids that are completely repeated four times and a fifth time partially. The second domain is adjacent to the first domain and is located on the /V-terminal side of it. It is composed of a single stretch of 43 amino acids. Protein F expressed in Escherichia coli completely blocked the binding of fibronectin to S. pyogenes. However, mutant proteins that contained only one or the other of the two domains were only capable of partial blockage of binding. Complete blockage of binding of fibronectin could be achieved when a protein extract containing the N-terminal domain was mixed in a binding reaction with a protein extract containing the C-terminal domain. Similarly, a purified recombinant protein containing the two domains only, blocked the binding completely. In contrast, a purified recombinant protein containing just the C-terminal domain, blocked the binding partially. A clone exclusively expressing the C-terminal domain, completely blocked the binding of the 30 kDa N-terminal fragment of fibronectin to S. pyogenes, whereas a clone expressing the N-terminal domain failed to block the binding of this FN fragment. Thus, the two FN-binding domains of protein F are necessary for maximal bacterial binding and act in concert to enhance the binding to fibronectin. The possibility that the two domains bind to two different regions on the fibronectin molecule is discussed. 相似文献
12.
Iron-saturated human transferrin was digested with either chymotrypsin or trypsin to produce C-lobe and N-lobe protein fragments. Individual protein fragments were purified by a combination of gel filtration and Concanavalin A affinity chromatographic procedures. The C-lobe and N-lobe fragments of human transferrin were then used in binding assays to assess their ability in binding to the bacterial transferrin receptors. Competitive binding assays demonstrated that the C-lobe fragment of human transferrin binds as well as intact human transferrin to bacterial transterrin receptors from Neisseria meningitidis, Neisseria gonorrhoeae and Haemophlius influenzae. Using isogenic mutants of N. meningitidis deficient in either of the transferrin-binding proteins (Tbps), we demonstrated that both transferrin-binding proteins were able to bind to the C-lobe fragment of human transferrin. 相似文献
13.
Filtered proteins including the low-molecular-weight protein lysozyme are reabsorbed by the proximal tubule via adsorptive endocytosis. This process starts with binding of the protein to the brush-border membrane. The binding of 125I-labelled egg-white lysozyme (EC 3.2.1.17) to isolated brush-border membranes of rat kidney and the effect of several low-molecular weight proteins on that binding was determined. The Scatchard plot revealed a one-component binding type with a dissociation constant of 5.3 μM and 53.0 nmol/mg membrane protein for the number of binding sites. The binding of the cationic lysozyme was inhibited competitively by the addition of cationic cytochrome c to the incubation medium, while the neutral myoglobin had no effect. The anionic β-lactoglobulin A inhibited the lysozyme binding in a noncompetitive manner. These data suggest that the binding takes place between positively charged groups of the protein molecule and negative sites on the brush-border membrane, and, the competition between the cationic cytochrome c and the cationic lysozyme for the binding sites may be responsible for the inhibitory effect of cytochrome c on renal lysozyme reabsorption. The binding step at the brush-border membrane appears to be cation-selective. 相似文献
14.
Martha Valiadi Maria Debora Iglesias‐Rodriguez 《The Journal of eukaryotic microbiology》2014,61(2):134-145
Dinoflagellate bioluminescence systems operate with or without a luciferin binding protein, representing two distinct modes of light production. However, the distribution, diversity, and evolution of the luciferin binding protein gene within bioluminescent dinoflagellates are not well known. We used PCR to detect and partially sequence this gene from the heterotrophic dinoflagellate Noctiluca scintillans and a group of ecologically important gonyaulacoid species. We report an additional luciferin binding protein gene in N. scintillans which is not attached to luciferase, further to its typical combined bioluminescence gene. This supports the hypothesis that a profound re‐organization of the bioluminescence system has taken place in this organism. We also show that the luciferin binding protein gene is present in the genera Ceratocorys, Gonyaulax, and Protoceratium, and is prevalent in bioluminescent species of Alexandrium. Therefore, this gene is an integral component of the standard molecular bioluminescence machinery in dinoflagellates. Nucleotide sequences showed high within‐strain variation among gene copies, revealing a highly diverse gene family comprising multiple gene types in some organisms. Phylogenetic analyses showed that, in some species, the evolution of the luciferin binding protein gene was different from the organism's general phylogenies, highlighting the complex evolutionary history of dinoflagellate bioluminescence systems. 相似文献
15.
Dagmar G?rtner Jochen Degenkolb Jürgen A. E. Ripperger Rudolf Allmansberger Wolfgang Hillen 《Molecular & general genetics : MGG》1992,232(3):415-422
Summary A crude protein extract of Bacillus subtilis W23 contains a sequence-specific DNA binding activity for the xyl operator as detected by the gel mobility shift assay. A xylR determinant encoded on a multicopy plasmid leads to increased expression of this binding activity. In situ footprinting analysis of the protein-DNA complex in a polyacrylamide gel shows that the xyl operator is sequence-specifically bound and protected from cleavage by copper-phenanthroline at 26 phosphodiester bonds on each strand. Quantitative competition assays for repressor binding reveal that a 25 by synthetic xyl operator cloned into a polylinker is bound with the same affinity as the operator in the wild-type xyl regulatory region. This confirms that no additional sites in the wild-type sequence contribute to repressor binding. The xyl operator consists of ten palindromic base pairs flanking five central non-palindromic base pairs. A mutational analysis shows that the sequence of the central base pairs contributes to recognition by the repressor protein and that the spacing of the palindromic elements is crucial for repressor binding. An operator half site is not bound by the repressor. In vivo and in vitro induction studies suggest that, of several structurally similar sugars, xylose is the only molecular inducer of the Xyl repressor. 相似文献
16.
Robert S. Keyes Ying Y. Caot Elisabeth V. Bobst John M. Rosenberg Albert M. Bobst 《Journal of biomolecular structure & dynamics》2013,31(2):163-172
Abstract A complex consisting of the EcoRI endonuclease site-specifically bound to spin-labeled DNA 26mers was prepared to provide a model system for studying possible conformational changes resulting from protein binding. EPR was used to monitor the mobility of the spin labels that were strategically placed in position 6, 9, or 11 with respect to the dyad axis of the 26mer. These positions are located within the flanking region on either side of the EcoRI hexamer binding site. This allows the monitoring of potential distal structural changes in the DNA helix caused by protein binding. The spectral line shapes indicate that the spin label closest to the EcoRI endonuclease binding site, i.e., in position 6, is most influenced by the binding event. The EPR data are analyzed according to a model that distinguishes between spectral effects due to a change in the hydrodynamic shape of the complex and those resulting from local variations in the spin-label mobility as characterized by a local order parameter S. S reflecting the motional restriction of the spin-labeled base is 0.20 ± 0.01 for all three oligomers as well as for the two complexes with the label in position 9 or 11, while the position 6 labeled complex yields S=0.25. To further evaluate the origin of the slightly larger EPR effect observed with position 6 labeled material, molecular dynamics (MD) simulations were used to explore the space accessible to the probes in positions 6, 9, and 11. MD results gave similar nitroxide trajectories for all three labeled 26mers in the absence or presence of EcoRI. Thus, the small position 6 effect is attributed to a structural distortion in the major groove of the DNA at this location possibly corresponding to a bend induced by protein binding. The observation that the spectral changes are small indicates the absence of any significant structural disruption being propagated along the helix as a result of protein binding. Also, the fact that the line shape of the 26mers did not change as expected from hydrodynamic theory in view of the significant increase in molecular volume upon protein binding suggests that there are additional relaxation processes involving the protein and nucleic acid. 相似文献
17.
Isolation of two novel myb-like genes from Arabidopsis and studies on the DNA-binding properties of their products 总被引:2,自引:0,他引:2
Two novel myb-like genes (atmyb6 and atmyb7) were isolated from an Arabidopsis thaliana cDNA library. The entire proteins or the Myb domains encoded by the genes were expressed as fusion proteins in Escherichia coli. The DNA-binding domain of the murine c-Myb was also expressed in the same way for use in comparative studies. The fusion proteins were examined for their DNA-binding activity using the animal c-Myb DNA-binding site (MBS) and the binding site of the maize P gene product (PBS). The Myb domain of Atmyb6 bound to PBS more efficiently than to MBS. Complete Atmyb6 and Atmyb7 proteins preferentially bound to PBS but not MBS. This suggests that the in vitro binding consensus sequences for both Atmyb6 and Atmyb7 are similar to PBS. The binding of the Myb domain of Atmyb6 to both PBS and MBS raises the possibility that the protein recognizes multiple sequences in vivo. The third α-helix and three adjacent amino acids in the third repeat (R3) of c-Myb were replaced with the analogous sequence of Atmyb6 to create a chimeric Myb protein. This chimeric protein bound to PBS with a low affinity but failed to bind to MBS. Thus the binding pattern of the chimeric Myb protein is similar to that of the Atmyb6. This result suggests that the last 20 amino acids in the R3 repeat of Atmyb6 play a major role in DNA-binding. 相似文献
18.
19.
Alternate frame folding (AFF) is a mechanism by which conformational change can be engineered into a protein. The protein structure switches from the wild‐type fold (N) to a circularly‐permuted fold (N′), or vice versa, in response to a signaling event such as ligand binding. Despite the fact that the two native states have similar structures, their interconversion involves folding and unfolding of large parts of the molecule. This rearrangement is reported by fluorescent groups whose relative proximities change as a result of the order–disorder transition. The nature of the conformational change is expected to be similar from protein to protein; thus, it may be possible to employ AFF as a general method to create optical biosensors. Toward that goal, we test basic aspects of the AFF mechanism using the AFF variant of calbindin D9k. A simple three‐state model for fold switching holds that N and N′ interconvert through the unfolded state. This model predicts that the fundamental properties of the switch—calcium binding affinity, signal response (i.e., fluorescence change upon binding), and switching rate—can be controlled by altering the relative stabilities of N and N′. We find that selectively destabilizing N or N′ changes the equilibrium properties of the switch (binding affinity and signal response) in accordance with the model. However, kinetic data indicate that the switching pathway does not require whole‐molecule unfolding. The rate is instead limited by unfolding of a portion of the protein, possibly in concert with folding of a corresponding region. Proteins 2010. © 2010 Wiley‐Liss, Inc. 相似文献
20.
Hossein Hashemzadeh-Gargari Joel M. Wierenga 《Archives of insect biochemistry and physiology》1989,10(1):57-71
In attempting to develop an octopamine (OA) receptor preparation with ready access to large amounts of tissue, we examined the binding of OA to membranes from the heads of white and red houseflies (Musca domestica L.). Binding was dependent on the presence of L-ascorbic acid in the medium. However, equilibrium was reached only over 24–36 h at 4°C and reversal of binding was also slow and incomplete. Scatchard analysis revealed at least two binding sites in the white-eyed housefly. A high-affinity site (Kd = 13.9 nM and Bmax = 3.9 pmol/mg protein) was present, but the majority of the binding had low affinity (Kd = 1130 nM and Bmax = 165 pmol/mg protein). Scatchard analysis revealed a low affinity in the red-eyed housefly (Kd = 240 nM and Bmax = 12 pmol/mg protein). Catecholamines were the best competitors for OA binding followed by phenolamines such as OA and synephrine. 5-Hydroxytryptamine was less effective. Phentolamine and mianserin, which are good antagonists of the ability of OA to stimulate adenylate cyclase in housefly head membranes, and formamidine and imidazolines, which are potent partial agonists of this adenylate cyclase, were poor competitors of OA binding. The slow kinetics, low affinity, large amount, and unconventional pharmacological profile of this binding is not congruent with it being a neuroreceptor. When the brain was dissected free from the head, less than 10% of the total specific binding of OA was found in the brain membrane fraction. This suggests that most of the binding of OA may be to cuticular sites that possibly are associated with the metabolism of catecholamines in cuticular synthesis. Thus, binding studies made with labeled catecholamines and phenolamines on insect tissues containing significant cuticular elements should be interpreted with caution. 相似文献