共查询到20条相似文献,搜索用时 15 毫秒
1.
Hattori T Umetsu M Nakanishi T Tsumoto K Ohara S Abe H Naito M Asano R Adschiri T Kumagai I 《Biochemical and biophysical research communications》2008,365(4):751-757
Quite recently, a few antibodies against bulk material surface have been selected from a human repertoire antibody library, and they are attracting immense interest in the bottom-up integration of nanomaterials. Here, we constructed antibody fragments with binding affinity and specificity for nonbiological inorganic material surfaces by grafting material-binding peptides into loops of the complementarity determining region (CDR) of antibodies. Loops were replaced by peptides with affinity for zinc oxide and silver material surfaces. Selection of CDR loop for replacement was critical to the functionalization of the grafted fragments; the grafting of material-binding peptide into the CDR2 loop functionalized the antibody fragments with the same affinity and selectivity as the peptides used. Structural insight on the scaffold fragment used implies that material-binding peptide should be grafted onto the most exposed CDR loop on scaffold fragment. We show that the CDR-grafting technique leads to a build-up creation of the antibody with affinity for nonbiological materials. 相似文献
2.
M Sugimoto H Mohri R A McClintock Z M Ruggeri 《The Journal of biological chemistry》1991,266(27):18172-18178
We have used proteolytic fragments and overlapping synthetic peptides to define the domain of von Willebrand factor (vWF) that forms a complex with botrocetin and modulates binding to platelet glycoprotein (GP) Ib. Both functions were inhibited by the dimeric 116-kDa tryptic fragment and by its constituent 52/48-kDa subunit, comprising residues 449-728 of mature vWF, but not by the dimeric fragment III-T2 which lacks amino acid residues 512-673. Three synthetic peptides, representing discrete discontinuous sequences within the region lacking in fragment III-T2, inhibited vWF-botrocetin complex formation; they corresponded to residues 539-553, 569-583, and 629-643. The 116-kDa domain, with intact disulfide bonds, exhibited greater affinity for botrocetin than did the reduced and alkylated 52/48-kDa molecule, and both fragments had significantly greater affinity than any of the inhibitory peptides. Thus, conformational attributes, though not strictly required for the interaction, contribute to the optimal functional assembly of the botrocetin-binding site. Accordingly, 125I-labeled botrocetin bound to vWF and to the 116-kDa fragment immobilized onto nitrocellulose but not to equivalent amounts of the reduced and alkylated 52/48-kDa fragment; it also bound to the peptide 539-553, but only when the peptide was immobilized onto nitrocellulose at a much greater concentration than vWF or the proteolytic fragments. These studies demonstrate that vWF interaction with GP Ib may be modulated by botrocetin binding to a discontinuous site located within residues 539-643. The finding that single point mutations in Type IIB von Willebrand disease are located in the same region of the molecule supports the concept that this domain may contain regulatory elements that modulate vWF affinity for platelets at sites of vascular injury. 相似文献
3.
van Dolleweerd CJ Kelly CG Chargelegue D Ma JK 《The Journal of biological chemistry》2004,279(21):22198-22203
Guy's 13 is a mouse monoclonal antibody that specifically recognizes the major cell-surface adhesion protein SA I/II of Streptococcus mutans, one of the major causative agents of dental caries. Passive immunization with Guy's 13 prevents bacterial colonization in humans. To help elucidate the mechanism of prevention of colonization conferred by this antibody, the SA I/II epitope recognized by Guy's 13 was investigated. It was previously established that the epitope is conformational, being assembled from two non-contiguous regions of SA I/II. In the current study, using recombinant fragments of SA I/II and, ultimately, synthetic peptides, the discontinuous epitope was localized to residues 170-218 and 956-969. This work describes the mapping of a novel discontinuous epitope that requires an interaction between each determinant in order for epitope assembly and recognition by antibody to take place. Guy's 13 binds to the assembled epitope but not to these individual epitope fragments. The assembled epitope results from the interaction between the individual antigenic determinants and can be formed by mixing together determinants present on separate polypeptide chains. The data are consistent with one of the epitope fragments adopting a polyproline II-like helical conformation. 相似文献
4.
Steffen van der Wal John A. W. Kruijtzer Rob M. J. Liskamp 《Journal of peptide science》2014,20(4):235-239
Synthetic mimics of discontinuous epitopes may have a wide range of potential applications, including synthetic vaccines and inhibition of protein–protein interactions. However, synthetic access to these relatively complex peptide molecular constructs is limited. This paper describes a versatile convergent strategy for the construction of protein mimics presenting three different cyclic peptides. Using an orthogonal alkyne protection strategy, peptide loops were introduced successively onto a triazacyclophane scaffold via Cu(I)‐catalyzed azide alkyne cycloaddition. This method provides rapid access to protein mimics requiring different peptide segments for their interaction and activity. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
5.
S Kliche L Stitz H Mangalam L Shi T Binz H Niemann T Briese W I Lipkin 《Journal of virology》1996,70(11):8133-8137
Borna disease virus infection is diagnosed by the presence of serum antibodies reactive with the major viral proteins, p40 and p23. Although p40 and p23 are unrelated in amino acid sequence structure, cross-reactive antibodies are described. Protein fragments and synthetic peptides were analyzed to characterize the specificities of antibodies to p23. Epitope mapping revealed eight continuous epitopes accessible on the surface of a predicted structural model for the monomeric and the disulfide-linked dimeric forms of p23. None of these epitopes was reactive with antibodies to p40. Cross-reactivity with monospecific sera and monoclonal antibodies to p40 was found for one discontinuous epitope located at the amino terminus of p23. 相似文献
6.
Ines S. Jaeger Ines Kretzschmar Jana Körner Armin A. Weiser Carsten C. Mahrenholz Ajish Potty Katerina Kourentzi Richard C. Willson Rudolf Volkmer Robert Preissner 《Journal of molecular recognition : JMR》2013,26(1):23-31
To perform their various functions, protein surfaces often have to interact with each other in a specific way. Usually, only parts of a protein are accessible and can act as binding sites. Because proteins consist of polypeptide chains that fold into complex three‐dimensional shapes, binding sites can be divided into two different types: linear sites that follow the primary amino acid sequence and discontinuous binding sites, which are made up of short peptide fragments that are adjacent in spatial proximity. Such discontinuous binding sites dominate protein–protein interactions, but are difficult to identify. To meet this challenge, we combined a computational, structure‐based approach and an experimental, high‐throughput method. SUPERFICIAL is a program that uses protein structures as input and generates peptide libraries to represent the protein's surface. A large number of the predicted peptides can be simultaneously synthesised applying the SPOT technology. The results of a binding assay subsequently help to elucidate protein–protein interactions; the approach is applicable to any kind of protein. The crystal structure of the complex of hen egg lysozyme with the well‐characterised murine IgG1 antibody HyHEL‐5 is available, and the complex is known to have a discontinuous binding site. Using SUPERFICIAL, the entire surface of lysozyme was translated into a peptide library that was synthesised on a cellulose membrane using the SPOT technology and tested against the HyHEL‐5 antibody. In this way, it was possible to identify two peptides (longest common sequence and peptide 19) that represented the discontinuous epitope of lysozyme. Copyright © 2012 John Wiley & Sons, Ltd. 相似文献
7.
The utilization of biomaterials such as proteins or peptides has recently been focused on as an attractive way to construct nanomaterials by “bottom-up” strategy. We focus on α-synuclein as a novel scaffold material for functional nanomaterial fabrication. This protein constructs an amyloid-like nanostructure by self-assembly under mild conditions. In this paper, we demonstrate nanomaterial fabrication by utilizing two peptide fragments of the non-amyloid-β component of Alzheimer’s disease amyloid region, which is the key region for α-synuclein fibrillation. One of these peptide fragments contains the sequence corresponding to residues 66–82 of wild-type α-synuclein, while the other contains the same region from the Val70Thr/Val71Thr mutant, whose character is drastically different. In this paper, we confirmed that these two peptides individually formed different rod-like structures. Moreover, these peptides modify the fibril nanostructure of full-length α-synuclein, and these effects depend on the peptide sequences. Therefore, we propose the combination of amyloid-forming protein, and its partial peptide fragments with some mutations have a potential for novel nanomaterial fabrication. 相似文献
8.
Proteolytic excision and in situ cyclization of a bioactive loop from an REI-VL presentation scaffold. 总被引:1,自引:1,他引:0
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Covalent cyclization of peptides is an important tool in structure-function analysis of bioactive peptides, because it constrains the molecule to enrich or exclude the receptor-bound conformation. Previously we described a 2-step procedure for cyclizing purified, native peptides in aqueous solution by reacting a Met or Lys side chain with an iodoacetylated N-terminus (Wood SJ, Wetzel R, 1992a, Int J Pept Protein Res 39:533-539). We show here that the cyclization reaction scheme can be extended to peptides excised from proteins by endo-LysC proteolysis, which generates fragments terminating with Lys. To illustrate the method, we used an immunoglobulin VL domain (REI-VL) with an RGD-containing sequence engineered into its CDR3 and flanked by Lys residues. This REI-VL/RGD hybrid displayed an IC50 of 24 nM for ligand competition at the platelet fibrinogen receptor alpha IIb beta 3. The RGD-containing peptide excised by endo-LysC from the REI-VL presentation scaffold exhibited an IC50 of about 50 nM, and the corresponding cyclized peptide, and IC50 of about 10 nM. Significantly, both the N alpha-acylation and the cyclization reactions occur efficiently even in the context of the other endo-LysC fragments of REI-VL, which suggests that the reaction may prove useful in converting mixtures of endo-LysC products of many proteins into the corresponding cyclic peptides in situ. 相似文献
9.
Peptide aptamers are peptides constrained and presented by a scaffold protein that are used to study protein function in cells. They are able to disrupt protein-protein interactions and to constitute recognition modules that allow the creation of a molecular toolkit for the intracellular analysis of protein function. The success of peptide aptamer technology is critically dependent on the performance of the scaffold. Here, we describe a rational approach to the design of a new peptide aptamer scaffold. We outline the qualities that an ideal scaffold would need to possess to be broadly useful for in vitro and in vivo studies and apply these criteria to the design of a new scaffold, called STM. Starting from the small, stable intracellular protease inhibitor stefin A, we have engineered a biologically neutral scaffold that retains the stable conformation of the parent protein. We show that STM is able to present peptides that bind to targets of interest, both in the context of known interactors and in library screens. Molecular tools based on our scaffold are likely to be used in a wide range of studies of biological pathways, and in the validation of drug targets. 相似文献
10.
Proteolytic digestion of proteins bound to immobilized antibodies, combined with matrix assisted laser desorption (MALDI)
mass spectrometric identification of the affinity-bound peptides, can be a powerful technique for epitope determination. Binding
of the protein to the antibody is done while the protein is in its native, folded state. A purified protein is not required
for this procedure, because only proteins containing the antigenic determinant will bind to the antibody in the initial step.
The method makes use of the resistance of the antibody to enzymatic digestion. Enzymatic cleavage products of the antigenic
protein not containing the epitope are washed off the beads, leaving the epitope-containing fragments affinity bound to the
immobilized antibody. Dissociation of the antigen-antibody complex prior to mass spectrometric analysis is unnecessary because
the affinity-bound peptides are released by the MALDI matrix crystallization process, although the antibody remains covalently
attached to the sepharose beads.
This epitope-mapping protocol has been used in the determination of both continuous and discontinuous epitopes on both glycosylated
and unglycosylated proteins. 相似文献
11.
A discontinuous factor H binding site in the third component of complement as delineated by synthetic peptides 总被引:2,自引:0,他引:2
J D Lambris D Avila J D Becherer H J Müller-Eberhard 《The Journal of biological chemistry》1988,263(24):12147-12150
Factor H, a very important regulator of alternative pathway activation, exerts its effects by binding to the third component complement, C3. In this study we present evidence that factor H reacts with at least two sites in the third component of complement (C3), and we have mapped one of these sites within the C3d fragment of C3. By using direct binding assays of an anti-human H anti-idiotypic antibody (alpha alpha H) and of H to C3 fragments, it was shown that both bound to the C3b and C3d (but not to C3c) fragments of C3. Cleavage of C3d by CNBr generated two major fragments with Mr values of 12,500 (residues 997-1107) and 8,600 (residues 1178-1252). Binding studies with these two fragments showed that only the Mr 8,600 fragment bound to both H and alpha alpha H. Several synthetic peptides (A58, 1192-1249; P28, 1187-1214; P16, 1194-1209; P14, 1201-1214; B17, 1206-1222; J28, 1222-1249; and J16, 1234-1249) were synthesized according to the primary sequence of the Mr 8,600 fragment. Based on the differential binding of these synthetic peptides to H, their inhibitory effect on H binding to C3b or C3d, and their effect on H cofactor activity, we mapped the H binding site in C3 to a discontinuous site spanning residues 1187-1249 of the C3 sequence. By studying the inhibition of H binding to C3b or C3d by the different synthetic peptides, we also present evidence that a second binding site in C3b for H exists. 相似文献
12.
N A Lokker U Strittmatter C Steiner B Fagg P Graff H P Kocher G Zenke 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(3):893-898
The epitopes of neutralizing mAb were mapped in order to identify a receptor binding site on human IL-3 (huIL-3). To initiate this structure and activity analysis, four neutralizing mAb were selected on the basis of preventing rhuIL-3 stimulated proliferation of peripheral blood cells from a patient with chronic myelogenous leukemia (CML). In order to identify continuous epitopes, the neutralizing mAb were assayed in a solid-phase ELISA for their reactivity with either denatured rhuIL-3 or with the peptides generated by digestion of rhuIL-3 by using two different proteinases. Two of the neutralizing mAb recognized single fragments from both digestions. Amino acid (aa) sequence determination showed that these peptides overlap, defining a region of 22 aa (aa 29 to 50 of the mature rhuIL-3 protein). In a competition ELISA, the two continuous epitopes were shown to be linked to one another and to the two discontinuous epitopes, suggesting that all four neutralizing mAb bind to a discrete region of the IL-3 molecule, which might be involved in binding to the IL-3R. 相似文献
13.
Target validation using protein aptamers enables the characterization of a specific function of a target protein in an environment that resembles native conditions as closely as possible. A major obstacle to the use of this technology has been the generation of bioactive aptamers, which is dependent on the choice of scaffold. Constraining binding peptides within a particular scaffold does not necessarily result in binding aptamers, as suboptimal presentation of peptides can occur. It is therefore understandable that different peptides might require different scaffolds for optimal presentation. In this article, we describe a novel scaffold protein that bypasses the conventional requirement for scaffolds to have known rigid structures and yet successfully presents several peptides that need to adopt a wide range of conformations for binding to their target protein. Using an unstructured protein, 4EBP1, as scaffold, we successfully construct binding aptamers to three different target proteins: Mdm2, proliferating cell nuclear antigen, and cyclin A. The Mdm2-binding aptamer constructed using 4EBP1 as scaffold demonstrates better stability and bioactivity compared to that constructed using thioredoxin as scaffold. This new scaffold protein, which makes it relatively easy to create bioactive aptamers based on known interaction sequences, will greatly facilitate the aptamer approach to target validation. 相似文献
14.
Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries 总被引:10,自引:0,他引:10
Söderlind E Strandberg L Jirholt P Kobayashi N Alexeiva V Aberg AM Nilsson A Jansson B Ohlin M Wingren C Danielsson L Carlsson R Borrebaeck CA 《Nature biotechnology》2000,18(8):852-856
We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 x 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules. 相似文献
15.
Lindman S Lindeberg G Nyberg F Karlén A Hallberg A 《Bioorganic & medicinal chemistry》2000,8(9):2375-2383
Rigidification of peptides by cyclization and iterative incorporation of well-defined secondary structure mimetics constitutes one approach to the design of non-peptidergic structures with better defined conformations. We herein present the synthesis of a potential gamma-turn mimetic scaffold, and its incorporation in the 3-5 position of angiotensin II. Two analogues of angiotensin II (Ang II) incorporating this 1,3,5-trisubstituted benzene gamma-turn scaffold were synthesized. Evaluation of the compounds in a radioligand binding assay showed that they lacked affinity to the AT1 receptor. To rationalize these results a geometrical and electrostatical comparison with Ang II analogues encompassing a bicyclic scaffold that delivered inactive pseudo peptides and an azepine scaffold producing highly active ligands was made. This analysis did not provide a clear rationale for the inactivity of the benzene gamma-turn scaffolds. 相似文献
16.
Skripnikov AIu Anikanov NA Kazakov VS Dolgov SV Ziganshin RKh Govorun VM Ivanov VT 《Bioorganicheskaia khimiia》2011,37(1):108-118
In the current study the isolation and identification of Physcomitrella patens (Hedw.) B.S.G. moss peptides are described. Physcomitrella patens moss is actively used in recent years as a model organism to study the biology of plants. Protoplasts, protonemata and gametophores of the moss are demonstrated for the first time to contain diverse small peptides. From gametophores was isolated and identified 58 peptides that are fragments of 14 proteins, and from protonemata - 49 peptides, fragments of 15 proteins. It was found that the protonemata and gametophores Ph. patens, which are the successive stages of development of this plant, significantly different from each other as a peptide composition and the spectrum of the precursor protein of identified peptides. Isolation of protoplasts of the enzymatic destruction of cell wall protonemata accompanied by massive degradation of intracellular proteins, many of whom are proteins of photosynthesis, which is a characteristic response of plants to stress the impact of environmental factors. A total of moss protoplasts were isolated and identified 323 peptides that are fragments of 79 proteins. 相似文献
17.
18.
Loop peptides stabilized by two β-strands were used as a scaffold for a phage displayed peptide library. Affinity-based screening for insulin provided peptides, which showed affinity constants of 10(5) M(-1) order for insulin over 100 times greater than their affinity for the structurally similar insulin-like growth factor 1. The results suggested that the scaffold offers a powerful tool for generating and screening peptides as ligands for drugs and biologics. 相似文献
19.
Bacillus subtilis bacteriophages SP82, SPO1, and phie: a comparison of DNAs and of peptides synthesized during infection. 总被引:5,自引:5,他引:0
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The genomes of Bacillus subtilis phages phie, SPO1, and SP82 were compared by DNA-DNA hybridization, analysis of DNA fragments produced by digestion with restriction endonucleases, comparison of the arrays of peptides synthesized during infection, and phage neutralization. DNA-DNA hybridization experiments indicated that about 78% of the SP82 DNA was homologous with SPO1 DNA, whereas 40% of the phie DNA was homologous to either SPO1 or SP82 DNA. Agarose gel electrophoresis was used to compare the molecular weights of DNA fragments produced by cleavage of SP82, SPO1, and phie DNAs with the restriction endonucleases Hae III, Sal I, Hpa II, and Hha I. Digestion of the DNAs with Hae III and Sal I produced only a few fragments, whereas digestion with Hpa II and Hha I yielded 29 to 40 fragments, depending on the DNA and the enzyme. Comparing the Hpa II fragments, 51% of the SP82 fragments had mobilities which matched those of SPO1 fragments, 32% of the SP82 fragments matched the phie fragments, and 34% of the SPO1 fragments matched the phie fragments. Comparing the Hha I digestion products, 62% of the SP82 fragments had mobilities matching the SPO1 fragments, 24% of the SP82 fragments matched the phie fragments, and 22% of the SPO1 fragments matched the phie fragments. Analysis of peptides by electrophoresis on one-dimensional sodium dodecyl sulfate-polyacrylamide slab gels showed that approximately 70 phage-specific peptides were synthesized in the first 24 min of each infection. With mobility and the intervals of synthesis as criteria, 66% of the different SP82 peptides matched the SPO1 peptides, 34% of the SP82 peptides matched the phie peptides, and 37% of the SPO1 peptides matched the phie peptides. Phage neutralization assays using antiserum to SP82 yielded K values of 510 for SP82, 240 for SPO1, and 120 for phie. 相似文献
20.
A computer program is designed to facilitate the identification of coding gene's fragments using a set of peptides. The program is written on Basic programming language for personal computer "Iskra-226" (USSR). To accelerate some operations, computer code commands are used. Treatment of 50 DNA fragments by means of 10 peptides takes ca. 1 h of computer time. The program outputs list coding gene's fragments and corresponding peptides. The suggested algorithm is based on our finding that the number of false identifications of a coding gene fragments may be predicted by Poisson distribution and minimized using correct criteria. The suggested method enables one to evaluate the reliability of the true identification of DNA fragments in case of mistakes in primary structure of the gene fragments or peptides. 相似文献