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Binding Specificity (binding + specificity)
Selected AbstractsBinding specificity and the ligand dissociation process in the E. coli biotin holoenzyme synthetasePROTEIN SCIENCE, Issue 3 2002Keehwan Kwon Abstract The binding of the Escherichia coli biotin holoenzyme synthetase to the two ligands, biotin and bio-5,-AMP, is coupled to disorder-to-order transitions in the protein. In the structure of the biotin complex, a "glycine-rich" loop that is disordered in the apo-enzyme is folded over the ligand. Mutations in three residues in this loop result in significant changes in the affinity of the enzyme for both biotin and bio-5,-AMP. The kinetic basis of these losses in the affinity resides primarily in changes in the unimolecular rates of dissociation of the complexes. In this work, isothermal titration calorimetry has been employed to examine the detailed thermodynamics of binding of three loop mutants to biotin and bio-5,-AMP. The energetic features of dissociation of the protein,ligand complexes also have been probed by measuring the temperature dependencies of the unimolecular dissociation rates. Analysis of the data using the Eyring formalism yielded entropic and enthalpic contributions to the energetic barrier to dissociation. The thermodynamic results coupled with the known structures of the apo-enzyme and biotin complex have been used to formulate a model for progression from the ground-state complex to the transition state in biotin dissociation. In this model, the transition-state is characterized by both partial disruption of noncovalent bonds and acquisition of some of the disorder that characterizes the glycine-rich loop in the absence of ligand. [source] Differential binding of endogenous steroids and chemicals to androgen receptors in rainbow trout and goldfishENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2000Kelly Wells Abstract Androgen receptors (ARs) from fish were characterized in order to evaluate differences in the binding affinities of steroids and environmental chemicals between mammals and fish, among species offish, and among target tissues within a species of fish. High-affinity, low-capacity ARs were identified in cytosolic fractions of rainbow trout brains (Oncorhynchus mykiss) and the brains, ovaries, and testes of goldfish (Carassius auratus) using [3H]testosterone. The binding specificities of endogenous steroids to the ARs did not differ between goldfish tissues but did differ between goldfish and rainbow trout. Interspecies differences in binding specificities were also seen using cyproterone acetate, which bound to the ARs in the goldfish tissues, but not in the rainbow trout brains. The mammalian antiandrogens flutamide, vinclozolin and its metabolites 2-(((3,5)-dichlorophenyl-carbamo-yl)oxy)-2-methyl-3-butenoic acid and 3,,5,-dichloro-2-hydroxy-2-methylbut-3-enanilide, along with procymidone did not bind to the ARs in any of the fish tissues tested. However, other mammalian antiandrogens including methoxychlor and its metabolite 2,2-bis(p -hydroxyphenyl)-1,1,1-trichloroethane, o,p,-DDT, o,p,-dichlorodiphenyldichloroethylene (DDE) and p,p,-DDE did bind to the fish ARs, but only in the goldfish testes, demonstrating tissue differences in AR binding specificities of environmental chemicals. These results may be due to the presence of multiple AR isoforms in the different fish species and tissues. This study supports the growing evidence of species differences in the potency and actions of endocrine-disrupting chemicals and suggests that multiple species need to be tested when screening the receptor binding ability of potential endocrine-disrupting chemicals. [source] MHC Class II epitope predictive algorithmsIMMUNOLOGY, Issue 3 2010Morten Nielsen Summary Major histocompatibility complex class II (MHC-II) molecules sample peptides from the extracellular space, allowing the immune system to detect the presence of foreign microbes from this compartment. To be able to predict the immune response to given pathogens, a number of methods have been developed to predict peptide,MHC binding. However, few methods other than the pioneering TEPITOPE/ProPred method have been developed for MHC-II. Despite recent progress in method development, the predictive performance for MHC-II remains significantly lower than what can be obtained for MHC-I. One reason for this is that the MHC-II molecule is open at both ends allowing binding of peptides extending out of the groove. The binding core of MHC-II-bound peptides is therefore not known a priori and the binding motif is hence not readily discernible. Recent progress has been obtained by including the flanking residues in the predictions. All attempts to make ab initio predictions based on protein structure have failed to reach predictive performances similar to those that can be obtained by data-driven methods. Thousands of different MHC-II alleles exist in humans. Recently developed pan-specific methods have been able to make reasonably accurate predictions for alleles that were not included in the training data. These methods can be used to define supertypes (clusters) of MHC-II alleles where alleles within each supertype have similar binding specificities. Furthermore, the pan-specific methods have been used to make a graphical atlas such as the MHCMotifviewer, which allows for visual comparison of specificities of different alleles. [source] A survey of the 2001 to 2005 quartz crystal microbalance biosensor literature: applications of acoustic physics to the analysis of biomolecular interactionsJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2007Matthew A. Cooper Abstract The widespread exploitation of biosensors in the analysis of molecular recognition has its origins in the mid-1990s following the release of commercial systems based on surface plasmon resonance (SPR). More recently, platforms based on piezoelectric acoustic sensors (principally ,bulk acoustic wave' (BAW), ,thickness shear mode' (TSM) sensors or ,quartz crystal microbalances' (QCM)), have been released that are driving the publication of a large number of papers analysing binding specificities, affinities, kinetics and conformational changes associated with a molecular recognition event. This article highlights salient theoretical and practical aspects of the technologies that underpin acoustic analysis, then reviews exemplary papers in key application areas involving small molecular weight ligands, carbohydrates, proteins, nucleic acids, viruses, bacteria, cells and lipidic and polymeric interfaces. Key differentiators between optical and acoustic sensing modalities are also reviewed. Copyright © 2007 John Wiley & Sons, Ltd. [source] Trigger Factor and DnaK possess overlapping substrate pools and binding specificitiesMOLECULAR MICROBIOLOGY, Issue 5 2003Elke Deuerling Summary Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, ,tig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37°C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted ,tig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities. [source] Chemical Synthesis of Triple-Labelled Three-Helix Bundle Binding Proteins for Specific Fluorescent Detection of Unlabelled ProteinCHEMBIOCHEM, Issue 6 2005Torun Engfeldt Abstract Site-specifically triple-labelled three-helix bundle affinity proteins (affibody molecules) have been produced by total chemical synthesis. The 58 aa affinity proteins were assembled on an automated peptide synthesizer, followed by manual on-resin incorporation of three different reporter groups. An orthogonal protection strategy was developed for the site-specific introduction of 5-(2-aminethylamino)-1-naphthalenesulfonic acid (EDANS) and 6-(7-nitrobenzofurazan-4-ylamino)-hexanoic acid (NBDX), constituting a donor/acceptor pair for fluorescence resonance energy transfer (FRET), and a biotin moiety, used for surface immobilization. Circular dichroism and biosensor studies of the synthetic proteins and their recombinant counterparts revealed that the synthetic proteins were folded and retained their binding specificities. The biotin-conjugated protein could be immobilized onto a streptavidin surface without loss of activity. The synthetic, doubly fluorescent-labelled affinity proteins were shown to function as fluorescent biosensors in an assay for the specific detection of unlabelled human IgG and IgA. [source] Complementary expression and heterophilic interactions between igLON family members neurotrimin and LAMPDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2002Orlando D. Gil Abstract Neurotrimin (Ntm) and the limbic system-associated membrane protein (LAMP) are members of the IgLON (LAMP, OBCAM, Ntm) family of glycorylphosphatidylinositol anchored neural cell adhesion molecules. We previously reported that LAMP and Ntm promote adhesion and neurite outgrowth via a homophilic mechanism, suggesting that these proteins promote the formation of specific neuronal circuits by homophilic interactions. In this report, we have further characterized the expression and binding specificity of Ntm. Using a newly generated monoclonal antibody to Ntm, we demonstrated that this protein is largely expressed in a complementary pattern to that of LAMP in the nervous system, with co-expression at a few sites. Ntm is expressed at high levels in sensory-motor cortex and, of particular note, is transiently expressed in neurons of cortical barrel fields and corresponding thalamic "barreloids." Binding of a recombinant, soluble form of Ntm to CHO cells expressing either Ntm or LAMP demonstrates that Ntm and LAMP interact both homophilically and heterophilically. In contrast to conventional growth-promoting activity of Ig superfamily members, LAMP strongly inhibits the outgrowth of Ntm-expressing dorsal root ganglion (DRG) neurons in a heterophilic manner. These anatomical and functional data support the concept that homophilic and heterophilic interactions between IgLON family members are likely to play a role in the specification of neuronal projections via growth promoting and inhibiting effects, respectively. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 190,204, 2002 [source] Design and engineering human forms of monoclonal antibodiesDRUG DEVELOPMENT RESEARCH, Issue 3 2004Manuel L. Penichet Abstract The antibody molecule has multiple properties that make it a key component of the immune response. These include its ability to recognize a vast array of different foreign substrates and to interact with and activate the host effector systems. Antibodies with defined specificities may serve as "magic bullets" for the diagnosis and therapy of multiple diseases. With the development of the hybridoma technology, it was possible to produce rodent (mouse or rat) monoclonal antibodies that are the product of a single clone of antibody producing cells and have only one antigen binding specificity. However, the therapeutic use of rodent monoclonals antibodies in humans is limited by their immunogenicity, short circulating half-life, and inability to efficiently trigger human effector mechanisms. However, it proved difficult to produce human monoclonal antibodies using the same methods. To address these problems genetic engineering and expression systems have instead been used to produce chimeric, humanized, and totally human antibodies as well as antibodies with novel structures and functional properties. In addition, the use of yeast and human artificial chromosome vectors for animal transgenesis has allowed the development of animal models that produce antigen specific antibodies that are totally human. As a consequence, recombinant antibody-based therapies are now used to treat a variety of clinical conditions including infectious diseases, inflammatory disorders, and cancer. This article summarizes and compares different strategies for designing and engineering human antibodies and their derivatives. Drug Dev. Res. 61:121,136, 2004. © 2004 Wiley-Liss, Inc. [source] Microscale characterization of the binding specificity and affinity of a monoclonal antisulfotyrosyl IgG antibodyELECTROPHORESIS, Issue 12 2008Klaus S. Lassen Dr. Abstract Sulfation is a potentially important post-translational modification of proteins and has been demonstrated in a number of polypeptides, notably in gastrointestinal hormones. In contrast to phosphorylation, however, the investigation of sulfation patterns in tissues and on purified proteins has been complicated by the absence of specific immunoreagents (antibodies) for this modification as well as the chemical lability of the sulfate group. Here, we investigate the properties of a novel mAb against sulfated tyrosyl groups (anti-Tyr(SO3H) antibody) using CE and a panel of sulfated and nonsulfated peptides and proteins. The data show that the anti-Tyr(SO3H) antibody is completely specific for compounds containing sulfated tyrosyls. Affinity electrophoresis experiments allowed us to estimate dissociation constants for sulfated hirudin fragment (56,65), gastrin-17, and cholecystokinin octapeptide (CCK8) in the 1,3,,M range. The affinity of the antibody toward complement 4 protein that contains three sulfotyrosines was analyzed by surface plasmon resonance technology and modeled according to a bivalent-binding model which yielded a Kd1 of 20.1,,M for the monovalent complex. The same binding was studied by CE and found to be in the micromolar scale albeit with some uncertainty due to complex separation patterns. The work illustrates the amount of information on antibody,antigen interactions that may be obtained with microelectrophoretic methods consuming minute quantities of material. Furthermore the specificity of this antibody could be confirmed in one operation using an array of sulfated and nonsulfated compounds. [source] Redox-regulated affinity of the third PDZ domain in the phosphotyrosine phosphatase PTP-BL for cysteine-containing target peptidesFEBS JOURNAL, Issue 13 2005Lieke C. J. Van Den Berk PDZ domains are protein,protein interaction modules that are crucial for the assembly of structural and signalling complexes. They specifically bind to short C-terminal peptides and occasionally to internal sequences that structurally resemble such peptide termini. The binding of PDZ domains is dominated by the residues at the P0 and P,2 position within these C-terminal targets, but other residues are also important in determining specificity. In this study, we analysed the binding specificity of the third PDZ domain of protein tyrosine phosphatase BAS-like (PTP-BL) using a C-terminal combinatorial peptide phage library. Binding of PDZ3 to C-termini is preferentially governed by two cysteine residues at the P,1 and P,4 position and a valine residue at the P0 position. Interestingly, we found that this binding is lost upon addition of the reducing agent dithiothrietol, indicating that the interaction is disulfide-bridge-dependent. Site-directed mutagenesis of the single cysteine residue in PDZ3 revealed that this bridge formation does not occur intermolecularly, between peptide and PDZ3 domain, but rather is intramolecular. These data point to a preference of PTP-BL PDZ3 for cyclic C-terminal targets, which may suggest a redox state-sensing role at the cell cortex. [source] Functional Hydrogel Surfaces: Binding Kinesin-Based Molecular Motor Proteins to Selected Patterned Sites,ADVANCED FUNCTIONAL MATERIALS, Issue 8 2005T. Yu Abstract Hydrogel microstructures with micrometer-scale topography and controllable functionality have great potential for numerous nanobiotechnology applications including, for example, three-dimensional structures that exhibit controlled interactions with proteins and cells. Taking advantage of the strong affinity of histidine (His) residues for metal-ion,nitrilotriacetic acid (NTA) complexes, we have chemically modified hydrogels to enable protein immobilization with retention of activity by incorporating 2-methacrylamidobutyl nitrilotriacetic acid, an NTA-containing monomer that can be copolymerized with a series of monomers to form NTA-containing hydrogels. By varying the NTA-monomer composition in the hydrogels, it is possible to control the amount of protein bound to the hydrogel surface. The retention of biological activity was demonstrated by microtubule gliding assays. Normally, hydrogels are resistant to protein binding, but we have selected these materials because of their porous nature. Bringing together hydrogel functionalization and soft-lithography patterning techniques, it was possible to create a hybrid hydrogel superstructure that possesses binding specificity to His-tagged protein in selected sites. This type of surface and microstructure is not only advantageous for motor protein integration, but it can also be generally applied to the formation of His-tagged molecules for sensors and biochip applications. [source] Study of peptide,sugar non-covalent complexes by infrared atmospheric pressure matrix-assisted laser desorption/ionizationJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 7 2004Christopher E. Von Seggern Abstract Infrared atmospheric pressure matrix-assisted laser desorption/ionization quadrupole ion trap mass spectrometry was applied to the study of siglec binding to oligosaccharide ligands. Peptides were designed to mimic the binding sites of three members of the siglec family: sialoadhesin, MAG and CD22. These peptides were tested for their ability to complex with their carbohydrate ligands 3, -sialyllactose (3,SL) and 6, -sialyllactose (6,SL). All peptides demonstrated the ability to bind to the carbohydrates, with the peptide representing sialoadhesin maintaining its binding specificity for 3,SL in preference to 6,SL. This technique can be used to study other protein,sugar interactions and can be expanded to create high-throughput screening techniques. Copyright © 2004 John Wiley & Sons, Ltd. [source] Structural compatibility of novel nucleotide modifications with shortened linkages designed for antigene/antisense therapyJOURNAL OF RAMAN SPECTROSCOPY, Issue 5 2004J. Hanu Abstract The impact of isopolar shortened internucleotide linkage modification on the hybridization properties of potential antisense or antigene oligonucleotides was studied by using a model molecular system consisting of polyuridylic acid (PolyU) and analogs of diadenosine monophosphate with the modified linkage. Raman spectra of mixed aqueous solutions were measured at various temperatures and compositions of the mixtures for four types of modified linkage and natural ApA (3,,5,) as a reference. Analysis of the spectral sets provided amounts of formed complexes and their Raman spectra. It has been found that as in the case of ApA (3,,5,), the studied ApA analogs form with PolyU triplex-like complexes containing a central pseudo-chain of closely arranged adenosine dimers. In the case of S - and R -configured 2,,5, and 3,,5, ApCOH A analogs, respectively, the amounts of complexes formed even exceed the ApA. This hybridization effectiveness is reached, however, by a more feasible prolongation of the pseudo-chain, while the stability constant for the initiation step is significantly lower. Raman spectra of the complexes showed that for both of the above analogs the structural compatibility with the natural nucleic acid chain is decreased, because of the distorted position of one adenine residue. This distortion influences the Watson,Crick hydrogen bonds. The two types of linkages may be suitable just for construction of longer antigene or antisense oligonucleotides with alternating lengthened and shortened linkages. There is, however, a warning of possible decreased binding specificity. Copyright © 2004 John Wiley & Sons, Ltd. [source] Split target specificity of ResT: a design for protein delivery, site selectivity and regulation of enzyme activity?MOLECULAR MICROBIOLOGY, Issue 3 2007Makkuni Jayaram Summary The ResT telomere resolvase is responsible for maintaining the hairpin telomeres that cap the linear chromosome and minichromosomes of Borrelia burgdorferi. This enzyme acts at the tandem telomere junctions present within circular dimers resulting from DNA replication. ResT mediates the transesterification steps of resolution using a constellation of active site residues similar to that found in tyrosine recombinases and type IB topoisomerases. By combining this reaction mechanism with a hairpin binding module in its N-terminal domain, ResT reduces a fused telomere dimer into two hairpin monomers. ResT displays a split DNA binding specificity, with the N- and C-terminal domains targeting distinct regions of the telomere. This bi-specificity in binding is likely to be important in protein delivery, substrate selection and regulation of enzyme activity. [source] Quantitative specificity-based display library screening identifies determinants of antibody-epitope binding specificity,PROTEIN SCIENCE, Issue 9 2009Sejal S. Hall Abstract Despite the critical importance of molecular specificity in bimolecular systems, in vitro display technologies have been applied extensively for affinity maturation of peptides and antibodies without explicitly measuring the specificity of the desired interaction. We devised a general strategy to measure, screen, and evolve specificity of protein ligand interactions analogous to widely used affinity maturation strategies. The specificity of binding to target and nontarget antibodies labeled with spectrally distinct fluorophores was measured simultaneously in protein mixtures via multiparameter flow cytometry, thereby enabling screening for high target antibody specificity. Isolated antibody specific ligands exhibited varying specificity, revealing critical amino acid determinants for target recognition and nontarget avoidance in complex mixtures. Molecular specificity in the mixture was further enhanced by quantitative directed evolution, yielding a family of epitopes exhibiting improved specificities equivalent, or superior to, the native peptide antigen to which the antibody was raised. Specificity screening simultaneously favored affinity, yielding ligands with three-fold improved affinity relative to the parent epitope. Quantitative specificity screening will be useful to screen, evolve, and characterize the specificity of protein and peptide interactions for molecular recognition applications. [source] Effects of Castration on the Expression of Neurotrophic Factors in the Vas Deferens and Accessory Genital Glands of the RatANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 2005N. Mirabella Introduction:, Neurotrophic factors constitute a group of growth factor families, which have important effects on survival and differentiation of neuronal cells. The neurotrophin family is composed of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin (NT)3 and NT 4/5. Neurotrophins act by means of high (TrkA, TrkB and TrkC) and low (p75) affinity receptors on numerous neuronal populations of central and peripheral nervous system. The family of glial derived neurotrophic factor (GDNF) includes, besides the GDNF, neurturin (NTN), persephin (PSP) and artemin (ART). They bind to a common receptor Ret, but the binding specificity is due to a group of proteins (GFR, 1,4). These factors show a trophic effect on dorsal ganglia, motor neurons and autonomic nervous system. The aim of the present study is to evaluate the expression of NGF, BDNF and GDNF in the vas deferens and accessory genital glands of normal and castrated rats. Methods:, Immunohistochemistry, enzyme linked immunoassay (ELISA), reverse transcriptase (RT)-polymerase chain reaction (PCR). Results and Discussion:, Immunoreactivity to NGF, BDNF and GDNF was observed in all the investigated tracts. Generally, this immunoreactivity seemed to increase in castrated rats. ELISA and RT-PCR were performed to evaluate the levels of BDNF protein and its mRNA. In the normals, the greatest concentration of BDNF was observed in the vesicular gland, the lowest in the prostate. In the castrated, the BDNF concentration significantly decreased in the vas deferens. Conversely, it increased in the vesicular gland and in the ventral and dorsal prostate. BDNF transcripts were detected in both normal and castrated rats. These results suggest that neurotrophic factors are produced by the vas deferens and accessory genital glands and, in normal conditions, they are downregulated by androgens. [source] Crystallization and preliminary X-ray diffraction analysis of mouse galectin-4 N-terminal carbohydrate recognition domain in complex with lactoseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2008Veronika Krej, íková Galectin-4 is thought to play a role in the process of tumour conversion of cells of the alimentary tract and the breast tissue; however, its exact function remains unknown. With the aim of elucidating the structural basis of mouse galectin-4 (mGal-4) binding specificity, we have undertaken X-ray analysis of the N-terminal domain, CRD1, of mGal-4 in complex with lactose (the basic building block of known galectin-4 carbohydrate ligands). Crystals of CRD1 in complex with lactose were obtained using vapour-diffusion techniques. The crystals belong to tetragonal space group P4212 with unit-cell parameters a = 91.1, b = 91.16, c = 57.10,Å and preliminary X-ray diffraction data were collected to 3.2,Å resolution. An optimized crystallization procedure and cryocooling protocol allowed us to extend resolution to 2.1,Å. Structure refinement is currently under way; the initial electron-density maps clearly show non-protein electron density in the vicinity of the carbohydrate binding site, indicating the presence of one lactose molecule. The structure will help to improve understanding of the binding specificity and function of the potential colon cancer marker galectin-4. [source] Glycochips from Polyanionic Glycopolymers as Tools for Detecting Shiga ToxinsCHEMBIOCHEM, Issue 17 2007Hirotaka Uzawa Dr. Abstract An alternating layer-by-layer adsorption methodology was applied to the assembly of glycochips by using synthetic polyanionic glycopolymers. Three glycochips carrying globobioside (Gb2), ,-lactoside (, -Lac), or , - D -mannoside (, -Man) residues were prepared, and used for the detection of Shiga toxins, Stx-1 and Stx-2, by using surface plasmon resonance (SPR). Using this method, we could confirm that both Stx-1 and Stx-2 show binding specificity for the Gb2 glycochip as well as a weak affinity for the , -Lac glycochip. The affinity constants of these toxins depended strongly on the sugar content of the Gb2 polymer used to prepare the glycochip. Greater affinity was observed for chips with a higher sugar content (up to 43,%) in the Gb2 glycopolymer. The maximal affinity constants of Stx-1 and Stx-2 (Ka=108,109,M,1) enabled highly sensitive and facile analysis (10 ng,mL,1, 30 min). When Gb2 glycopolymers were used as competitors, Stx-1 and Stx-2 behaved differently from one another in terms of their SPR response; this allowed us to perform discriminative analysis between the two toxins. [source] | ||||||||||||||||