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1.
It has recently been proposed that the role of neuroglobin in the protection of neurons from ischaemia induced cell death requires the formation of a transient complex with cytochrome c. No such complex has yet been isolated. Here, we present the results of soft docking calculations, which indicate one major binding site for cytochrome c to neuroglobin. The results yield a plausible structure for the most likely complex structure in which the hemes of each protein are in close contact. NMR analysis identifies the formation of a weak complex in which the heme group of cytochrome c is involved. surface plasmon resonance studies provide a value of 45muM for the equilibrium constant for cytochrome c binding to neuroglobin, which increases significantly as the ionic strength of the solution increases. The temperature dependence of the binding constant indicates that the complex formation is associated with a small unfavourable enthalpy change (1.9kcalmol(-1)) and a moderately large, favourable entropy change (14.8calmol(-1)deg(-1)). The sensitivity of the binding constant to the presence of salt suggests that the complex formation involves electrostatic interactions.  相似文献   

2.
The ability of apolipoprotein E (apoE) to bind to cell-surface glycosaminoglycans (GAGs) is important for lipoprotein remnant catabolism. Using surface plasmon resonance, we previously showed that the binding of apoE to heparin is a two-step process; the initial binding involves fast electrostatic interaction, followed by a slower hydrophobic interaction. Here we examined the contributions of the N- and C-terminal domains to each step of the binding of apoE isoforms to heparan sulfate (HS) and dermatan sulfate (DS). ApoE3 bound to less sulfated HS and DS with a decreased favorable free energy of binding in the first step compared to heparin, indicating that the degree of sulfation has a major effect on the electrostatic interaction of GAGs with apoE. Mutation of a key Lys residue in the N-terminal heparin binding site of apoE significantly affected this electrostatic interaction. Progressive truncation of the C-terminal alpha-helical regions which favors the monomeric form of apoE3 greatly weakened the ability of apoE3 to bind to HS, with a much reduced favorable free energy of binding of the first step, suggesting that the C-terminal domain contributes to the GAG binding of apoE by the oligomerization effect. In agreement with this, dimerization of the apoE3 N-terminal fragment via disulfide linkage restored the electrostatic interaction of apoE with HS. Significantly, apoE4 exhibited much stronger binding to HS and DS than apoE2 or apoE3 in both lipid-free and lipidated states, perhaps resulting from enhanced electrostatic interaction through the N-terminal domain. This isoform difference in GAG binding of apoE may be physiologically significant such as in the retention of apoE-containing lipoproteins in the arterial wall.  相似文献   

3.
Garai K  Baban B  Frieden C 《Biochemistry》2011,50(13):2550-2558
The apolipoprotein apoE plays a key role in cholesterol and lipid metabolism. There are three isoforms of this protein, one of which, apoE4, is the major risk factor for Alzheimer's disease. At micromolar concentrations all lipid-free apoE isoforms exist primarily as monomers, dimers, and tetramers. However, the molecular weight form of apoE that binds to lipid has not been clearly defined. We have examined the role of self-association of apoE with respect to interactions with phospholipids. Measurements of the time dependence of turbidity clearance of small unilamellar vesicles of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) upon addition of apoE show that higher molecular weight oligomers bind poorly if at all. The kinetic data can be described by a reaction model in which tetramers and dimers of apoE must first dissociate to monomers which then bind to the liposome surface in a fast and reversible manner. A slow but not readily reversible conformational conversion of the monomer then occurs. Prior knowledge of the rate constants for the association-dissociation process allows us to determine the rate constant of the conformational conversion. This rate constant is isoform dependent and appears to correlate with the stability of the apoE isoforms with the rate of dissociation of the apoE oligomers to monomers being the rate-limiting process for lipidation. Differences in the lipidation kinetics between the apoE isoforms arise from their differences in the self-association behavior leading to the conclusion that self-association behavior may influence biological functions of apoE in an isoform-dependent manner.  相似文献   

4.
The interactions between an oligomeric heat-shock protein, alpha-crystallin, and its individual subunits with unfolded proteins were monitored by surface plasmon resonance. Immobilization at the sensor chip allowed us for the first time to study isolated alpha-crystallin subunits under physiological conditions. We observe that these subunits, in contrast to alpha-crystallin oligomers, do not bind unfolded protein. Our data indicate that quaternary structure of alpha-crystallin is necessary for its chaperone-like activity.  相似文献   

5.
Tetrapyridylporphyrins containing four chloro(2,2′-bipyridine)platinum(II) complexes attached at the meta (3-H2TPtPyP) and para (4-H2TPtPyP) positions of the peripheral pyridine ligands were synthesized and their interaction with DNA investigated. The compounds were isolated in the solid state and characterized by means of spectroscopic and analytical techniques. According to molecular simulations, the two isomers exhibit contrasting structural characteristics, consistent with a saddle shape configuration for 3-H2TPtPyP and a planar geometry for 4-H2TPtPyP. Surface plasmon resonance studies were carried out on the interaction of the complexes with calf thymus DNA, revealing a preferential binding of 3-H2TPtPyP, presumably at the DNA major grooves.  相似文献   

6.
A self-assembled 3D hydrogel-nanoparticle composite integrated surface plasmon resonance (SPR) sensor is reported here. The novel assembled substrate was developed by means of a surface mediated radical co-polymerization process to obtain a highly sensitive hydrogel-based thin film that possesses specific binding sites for target analytes. Initially, amino group modified gold nanoparticles (AuNPs) were covalently linked to acrylic acid monomer. Following this, N-isopropylacrylamide (NIPAAm) and AuNPs linked acrylic acid (AAc) monomers were randomly co-polymerized by the "grafting from" method in the presence of initiator and crosslinker onto the sensing surface. Surface characterization techniques were utilized to evaluate the thickness and composition of the hydrogel-nanoparticle film. The sensing platform was employed to study the binding kinetics and conformational changes of the ArsA ATPase as a consequence of binding trivalent arsenicals under a variety of conditions. ArsA, the catalytic subunit of the ArsAB arsenite (As(III)) translocating ATPase, is one of the five proteins encoded by the arsenical resistance (ars) operon of plasmid R773 in cells of Escherichia coli, that confers resistance to trivalent and pentavalent salts of the metalloid arsenic. SPR measurements indicate that the 3D hydrogel-nanoparticle coated sensors exhibited a higher sensitivity than that of the 2D AuNPs decorated sensors. Binding of As(III) to ArsA is greatly facilitated by the presence of magnesium ion and ATP.  相似文献   

7.
This review describes a comprehensive analysis of a surface plasmon resonance (SPR)-based biosensor study of molecular interactions in the insulin-like growth factor (IGF) molecular axis. In this study, we focus on the interaction between the polypeptide growth factors IGF-I and IGF-II with six soluble IGF binding proteins (IGFBP 1-6), which occur naturally in various biological fluids. We have describe the conditions required for the accurate determination of kinetic rate constants for these interactions and highlight the experimental and theoretical pitfalls, which may be encountered in the early stages of such a study. We focus on IGFBP-5 and describe a site-directed mutagenesis study, which examines the contribution of various residues in the protein to high affinity interaction with IGF-I and -II. We analyse the interaction of IGFBP-5 (and IGFBP-3) with heparin and other biomolecules and describe experiments, which were designed to monitor multi-protein complex formation in this molecular axis.  相似文献   

8.
Nitrilotriacetic acid has been routinely used in protein purification for its high affinity for His-tagged protein in the presence of Ni2+. Here we reported a type of nitrilotriacetic acid chip (NTA-chip) prepared by transferring NTA-DOGS containing a lipid monolayer to a 50 nm thick gold layer deposited on a glass slide. The surface binding ability of His-tagged protein and regeneration of NTA chip were characterized using a synthetic polypeptide P1 (His-His-His-His-His-His--aminohexanoic-Gly-Gly-Arg-Gly-Asp-Ser). The effect of divalent cations on integrin binding affinity for RGD ligand was investigated after P1 had been immobilized onto the sensor chip. The results show that the NTA-chip is a useful tool to immobilize His-tagged protein on the chip surface, and can provide a functional orientation for further investigation. The results also show that removing of Ca2+ bound on low affinity sites or adding of Mn2+ can increase the binding ability of integrin.  相似文献   

9.
PDC-109, the major protein of bovine seminal plasma, binds to sperm plasma membranes upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. The binding process is mediated primarily by the specific interaction of PDC-109 with choline-containing phospholipids. In the present study the kinetics and mechanism of the interaction of PDC-109 with phospholipid membranes were investigated by the surface plasmon resonance technique. Binding of PDC-109 to different phospholipid membranes containing 20% cholesterol (wt/wt) indicated that binding occurs by a single-step mechanism. The association rate constant (k(1)) for the binding of PDC-109 to dimyristoylphosphatidylcholine (DMPC) membranes containing cholesterol was estimated to be 5.7 x 10(5) M(-1) s(-1) at 20 degrees C, while the values of k(1) estimated at the same temperature for the binding to membranes of negatively charged phospholipids such as dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidic acid (DMPA) containing 20% cholesterol (wt/wt) were at least three orders of magnitude lower. The dissociation rate constant (k(-1)) for the DMPC/PDC-109 system was found to be 2.7 x 10(-2) s(-1) whereas the k(-1) values obtained with DMPG and DMPA was about three to four times higher. From the kinetic data, the association constant for the binding of PDC-109 to DMPC was estimated as 2.1 x 10(7) M(-1). The association constants for different phospholipids investigated decrease in the order: DMPC > DMPG > DMPA > DMPE. Thus the higher affinity of PDC-109 for choline phospholipids is reflected in a faster association rate constant and a slower dissociation rate constant for DMPC as compared to the other phospholipids. Binding of PDC-109 to dimyristoylphosphatidylethanolamine and dipalmitoylphosphatidylethanolamine, which are also zwitterionic, was found to be very weak, clearly indicating that the charge on the lipid headgroup is not the determining factor for the binding. Analysis of the activation parameters indicates that the interaction of PDC-109 with DMPC membranes is favored by a strong entropic contribution, whereas negative entropic contribution is primarily responsible for the rather weak interaction of this protein with DMPA and DMPG.  相似文献   

10.
In this study, we fabricated a novel variable wavelength surface plasmon resonance (SPR) sensor, which detects resonance conditions such as a maximum attenuation wavelength, measuring change of microscopic refractive index. Such a change was measured to detect a salmonella antigen–antibody reaction and a penicillinase–penicillin reaction. Our experiments were performed after immobilizing a salmonella antibody on the sensor chip. We measured the shift in resonant wavelength during the antigen–antibody reaction for 30 min by injecting 5 × 107 cells/ml concentration of salmonella antigen solution into the sample chamber. Also, after immobilizing penicillinase on the sensor chip, we measured the shift in resonant wavelength during the reaction. Penicillin solution at 10 mM was injected into the sample chamber. The shift of resonant wavelength for each experiment was measured using a white light source, multimode optical fiber, a part of sensor chip and an optical spectrum analyzer.As a result, the resonant wavelength shifted about 0.26 nm/min owing to the salmonella antibody–antigen reaction. Thus, we could detect the change in wavelength (0.8 nm/min) through the interaction of penicillin and penicillinase for 15 min using variable wavelength SPR sensor.  相似文献   

11.
12.
We have developed a simple assay method for the evaluation of estrogen receptor (ER) binding capacity of chemicals without the use of radio- or fluorescence-labeled compounds. We used the solution competition assay by the BIACORE biosensor, a surface plasmon resonance biosensor, with estradiol as a ligand, human recombinant ER(alpha) (hrER(alpha)) as a high molecular weight (hmw) interactant and test chemicals as analytes. For the ligand, aminated estradiol with a spacer molecule (E2-17PeNH) was synthesized and immobilized on a carboxymethyl dextran-coated sensor chip by the amine coupling method. The injection of the hmw interactant hrER(alpha) to the biosensor raised the sensorgram, indicating its binding to the ligand E2-17PeNH. The binding of test chemicals to hrERalpha was determined as a reduction in the hrER(alpha) binding to E2-17PeNH. The dissociation constant for the binding to hrER(alpha) was calculated for estrone (4.29 x 10(-9)M), estradiol (4.04 x 10(-10)M), estriol (8.35 x 10(-10)M), tamoxifen (2.16 x 10(-8)M), diethylstilbestrol (1.46 x 10(-10)M), bisphenol A (1.35 x 10(-6)M) and 4-nonylphenol (7.49 x 10(-6)M), by plotting the data according to an equation based on mass action law. This method can also be used as a high throughput screening method.  相似文献   

13.
Claudin family transmembrane proteins play an important role in tight junction structure and function in epithelial cells. Among the 24 isoforms identified in mice and humans, claudin-4 and -3 serve as the receptor for Clostridium perfringens enterotoxin (Cpe). The second extracellular loop (Ecl2) of claudin-4 is responsible for the binding to the C-terminal 30 amino acids of Cpe (Cpe30). To define the structural constraints for the claudin-4/Cpe30 interaction, a surface plasmon resonance (SPR) method was developed. GST fusions with claudin-4 revealed that Ecl2 with the downstream transmembrane domain of claudin-4 reconstituted the basic structural requirement for optimal binding activity to Cpe30, with affinity in the nanomolar range. Two 12-mer peptides selected by phage display against claudin-4-transfected CHO cells and a 12-mer Cpe mutant peptide also showed significant affinity for claudin-4 with this SPR assay, suggesting that a short peptide can establish stable contact with Ecl2 with nanomolar affinity. Alignment of these short peptides unveiled a common Ecl2 binding motif: . Whereas the short peptides bound native claudin-4 on transfected CHO cells in pull-down assays, only the larger Cpe30 peptide affected trans-epithelial electrical resistance (TER) in peptide-treated Caco-2BBe monolayers. Importantly, Cpe30 retained its binding to claudin-4 when fused to the C terminus of influenza hemagglutinin, demonstrating that its binding activity can be maintained in a different biochemical context. These studies may help in the design of assays for membrane receptor interactions with soluble ligands, and in applying new targeting ligands to delivering attached "cargo" proteins.  相似文献   

14.
There is a growing demand for the development of a new bioanalytical technique that is capable of monitoring neuronal differentiation noninvasively, in real time, and without any fluorescent probes. In a previous article, we demonstrated that a high-resolution two-dimensional surface plasmon resonance (2D–SPR) imager was very useful to monitor cell response on chemical stimulation in which protein kinase C (PKC) translocation was related. In the current study, we focused on developing a new method for monitoring neuronal differentiation and examined the application of the high-resolution 2D–SPR imager to monitor neuronal differentiation noninvasively and by a label-free format. We successfully monitored the intracellular signal transduction, which was mainly translocation of PKC in PC12 cells by the 2D–SPR imager, and found that the cells treated with a differentiation factor, nerve growth factor (NGF), showed a remarkable enhancement of 2D–SPR response to muscarine, carbachol, and acetylcholine stimulation. The results demonstrated that 2D–SPR sensing is applicable to in situ assessment of neuronal differentiation and to studying the expression state of the specific receptors in the living state.  相似文献   

15.
Lipoprotein(a) [Lp(a)], but not low-density lipoprotein (LDL), was previously shown to impair the generation of fibrin-bound plasmin [Rouy et al. (1991) Arterioscler. Thromb. 11, 629-638] by a mechanism involving binding of Lp(a) to fibrin. It was therefore suggested that the binding was mediated by apolipoprotein(a) [apo(a)], a glycoprotein absent from LDL which has a high degree of homology with plasminogen, the precursor of the fibrinolytic enzyme plasmin. Here we have evaluated this hypothesis by performing comparative fibrin binding studies using a recombinant form of apo(a) containing 17 copies of the apo(a) domain resembling kringle 4 of plasminogen, native Lp(a), and Glu-plasminogen (Glu1-Asn791). Attempts were also made to identify the kringle domains involved in such interactions using isolated elastase-derived plasminogen fragments. The binding experiments were performed using a well-characterized model of an intact and of a plasmin-digested fibrin surface as described by Fleury and Anglés-Cano [(1991) Biochemistry 30, 7630-7638]. Binding of r-apo(a) to the fibrin surfaces was of high affinity (Kd = 26 +/- 8.4 nM for intact fibrin and 7.7 +/- 4.6 nM for plasmin-degraded fibrin) and obeyed the Langmuir equation for adsorption at interfaces. The binding to both surfaces was inhibited by the lysine analogue AMCHA and was completely abolished upon treatment of the degraded surface with carboxypeptidase B, indicating that r-apo(a) binds to both the intrachain lysines of intact fibrin and the carboxy-terminal lysines of degraded fibrin. As expected from these results, both r-apo(a) and native Lp(a) inhibited the binding of Glu-plasminogen to the fibrin surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

16.
In this paper, a new simple approach for sensitivity optimization in surface plasmon resonance (SPR) chemosensors based on colorimetric ligands is presented. A new design of SPR sensor with tunable analytical wavelength (lambda(SPR)) was constructed for this purpose, to perform studies on the ligand absorbance spectra related sensitivity enhancement. Unlike commercial SPR sensors which operate at one lambda(SPR), the new device can be used for sensitivity analysis at selected lambda(SPR) in the range 550-750 nm, offering the possibility to identify the highest sensitivity lambda(SPR) in regard to the spectral changes of the selected ligand. Measurements can be easily done in ligand bulk solutions without immobilization steps. Sensitivity enhancement analysis and optimization of lambda(SPR) on chromogenic reagents with hypsochromic shift in their absorption spectra are demonstrated in this contribution. Optimal selection of analytical wavelength, set at the absorbance peak of chromogenic reagent Eriochrome Black T (EBT) was observed to result in up to two times increased SPR sensitivity to Cd(2+) compared to wavelengths selected in other parts of the ligand absorbance spectra, with a limit of detection (LOD) 0.2 ppm. The sensitivity enhancement at optimal lambda(SPR) was observed to be related to increased refractive index (n), drop in extinction coefficient (alpha) and simultaneous hypsochromic shift of the EBT absorbance spectra causing the lambda(SPR) to match the absorbance peak shoulder.  相似文献   

17.
T Valdes-Gonzalez  J Inagawa  T Ido 《Peptides》2001,22(7):1099-1106
Using Surface Plasmon Resonance (SPR) we investigated the interaction of seven neuropeptides with different characteristics and beta-amyloid (Abeta42) peptide, with membranes containing gangliosides. A wide range of affinities characterized the bindings (K(D) = 10(-3)- 10(-7) M), following the scheme: for GM1, Abeta42 > DYN > SP = GAL = SOM = BRD > OXY = ENK; for GD1a, Abeta42 = DYN = GAL > SP = SOM = BRD = OXY > ENK and for GT1b, Abeta42 > DYN > SP = GAL > SOM = BRD = OXY > ENK. The ganglioside sugar moiety, specifically the sialic acid, had an important role in the interactions. In general the affinities were higher with polysialo, than with monosialo gangliosides. The sensorgrams describing the interactions of Abeta42 and SP with gangliosides differed from the interactions of the other studied peptides. Ca(2+) promoted changes in peptide-glycolipid interactions.  相似文献   

18.
We have used surface plasmon resonance to investigate the nucleic acid binding properties of the core protein of hepatitis C virus, a disordered protein believed to chaperone the genomic RNA. It was previously shown that a peptide (peptide E) corresponding to the association of two basic clusters of core enhances the annealing and the dimerization of nucleic acid fragments derived from a stem loop (SL2) in the 3′ untranslated region of the hepatitis C virus genome. However, strong aggregation of nucleic acids by core or peptide E in the excess of the latter precluded the characterization of their binding parameters up to now. By careful design of surface plasmon resonance experiments, we obtained accurate binding parameters for the interaction of peptide E with SL2-derived oligonucleotides of different lengths and sequences, in form of stem-loop, duplex or strand. Peptide E was found to bind in a salt dependent manner to all oligonucleotides assayed. Affinity data identify at least two binding modes, of which one is independent of sequence/structure, and the other is specific to the SL2 stem-loop fold. Stoichiometry data support a multi-motif binding model allowing formation of higher-order complexes. We propose that the modular binding mode demonstrated for structured RNA-binding proteins also applies to this disordered chaperone and is relevant to its activity.  相似文献   

19.
Electroelution is a widely used methodology for protein purification. In this study, a practical and low-cost system for protein electroelution from stained polyacrylamide gels was developed. For this, a horizontal protein electroelution cuve was constructed with glass plates, 1.5-ml capacity microcentrifuge tubes, and dialysis membrane. Analyses of the system efficiency showed high protein recovery from nonfixed and fixed sodium dodecyl sulfate (SDS)-polyacrylamide gels.  相似文献   

20.
A new binding assay to investigate the mechanism of adhesion of lactic acid bacteria to the human intestine was established by the surface plasmon resonance technique using a biosensor BIACORE1000. Cells of 26 strains of the Lactobacillus acidophilus group as analytes were eluted onto a sensor chip on which were immobilized biotinylated A-trisaccharide polymer probes having human A-type antigen [(GalNAcalpha1-3(Fucalpha1-2)Gal)-] or human colonic mucin of blood type A (HCM-A) as ligands. In the first screening, high adhesive affinity to the A-trisaccharide BP-probe was observed in L. acidophilus OLL2769, L. crispatus JCM8778, LA205 and LA206. In the second screening, which used HCM-A, only L. acidophilus OLL2769 and L. crispatus JCM8778 were selected as adhesive strains with specific binding ability to human A-antigen. The results indicated that some strains of the L. acidophilus group could recognize and bind the sugar chain of A-antigen structure on HCM.  相似文献   

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