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  • 1
    Abstract: 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.
    Type of Publication: Journal article published
    PubMed ID: 23280614
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  • 2
    Keywords: Germany ; INFORMATION ; SUPPORT ; GENE-EXPRESSION ; GENOME ; microarray ; PROTEIN ; PROTEINS ; MOLECULES ; COMPLEX ; COMPLEXES ; SEQUENCE ; MOLECULE ; ACID ; ACIDS ; antibodies ; antibody ; NUCLEIC-ACID ; NUCLEIC-ACIDS ; LTD ; EQUIVALENT ; ASSAY ; microarrays ; ARRAYS ; NUMBER ; SURFACE ; DIVERSITY ; sensitivity ; ATOMIC-FORCE MICROSCOPY ; CONSTRUCTION ; expression profiling ; glass slide ; IMMUNOASSAYS ; LINKED-IMMUNOSORBENT-ASSAY ; surface modification ; ANTIBODY IMMOBILIZATION ; ELECTROSPRAY DEPOSITION ; immunoassay ; METAL-OXIDE SURFACES ; ORIENTED IMMOBILIZATION ; POLY(ETHYLENE GLYCOL) ; POLY(L-LYSINE)-G-POLY(ETHYLENE GLYCOL) LAYERS ; protein interaction ; protein microarray ; PROTEIN MICROARRAYS ; surface chemistry
    Abstract: Stimulated by the achievements of the first phase in genomics and the resulting need of assigning functions to the acquired sequence information, novel formats of immunoassays are being developed for high-throughput multi-analyte studies. In principle, they are similar in nature to the microarray assays already established at the level of nucleic acids. However, the biochemical diversity and the sheer number of proteins are such that an equivalent analysis is much more complex and thus difficult to accomplish. The wide range of protein concentration complicates matters further. Performing microarray immunoassays already represents a challenge at the level of preparing a working chip surface. Arrays have been produced on filter supports, in microtiter plate wells and on glass slides, the last two usually coated with one-, two- or three-dimensionally structured surface modifications. The usefulness and suitability of all these support media for the construction and application of antibody microarrays are reviewed in this manuscript in terms of the different kinds of immunoassay and the various detection procedures. Additionally, the employment of microarrays containing alternative sensor molecules is discussed in this context. The sensitivity of microspot immunoassays predicted by the current analyte theory is not yet a reality, indicating the extent of both the technology's potential and the size of the task still ahead. Copyright (C) 2003 John Wiley Sons, Ltd
    Type of Publication: Journal article published
    PubMed ID: 12898667
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  • 3
    Keywords: PEPTIDE ; CELL ; Germany ; IN-VIVO ; DISEASE ; DISEASES ; GENE ; GENE-EXPRESSION ; PROTEIN ; transcription ; DRUG ; MOLECULES ; CONTRAST ; treatment ; DISCOVERY ; TRANSPORT ; MEMBRANE ; MODULATION ; NUCLEUS ; PEPTIDES ; TRANSFORMATION ; HUMAN-PAPILLOMAVIRUS ; MALIGNANT TRANSFORMATION ; C-MYC ; CHRONIC MYELOGENOUS LEUKEMIA ; CHRONIC MYELOID-LEUKEMIA ; DRUG-RESISTANCE ; ANTISENSE OLIGONUCLEOTIDES ; drug design,drug delivery,membrane transport,NLS,cancer treatment,radiation,peptide synthesis,solid- ; HUMAN PROSTATE-CANCER ; NEUTRON-CAPTURE THERAPY ; nuclear envelope ; NUCLEAR-ENVELOPE
    Abstract: The unique functions of biomolecules, including transport accross biological membranes (e.g. the cell membrane, the nuclear envelope), modulation of protein function, gene transcription, reconstitution of the malignant transformation, and viral, bacterial and fungal activities underlie a high pharmaceutical potential. The development of combinatorial functional peptide modules in this important area has been slow, in contrast to the rapid development in the synthesis of small biopolymers. The conjugation of a short transmembrane transport peptide module with a cell nucleus address peptide module and with any substance is attractive for preparation of BioShuttle-based peptides because of the well-established automated synthesis of peptides. Variation of the different functional modules for drug targeting and the choice of substances can be combined to create novel bioconjugates with unique properties. This article provides an overview of previous work on the BioShuttle technology and outlines the promising use of this approach in combinatorial peptide synthesis and drug discovery. Copyright (C) 2003 John Wiley Sons, Ltd
    Type of Publication: Journal article published
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  • 4
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 1 (1988), S. 32-41 
    ISSN: 0952-3499
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We sought to identify the features controlling the specificity of antibody recognition and thus gain insights into molecular recognition between proteins in general. A total of 103 epitopes with in 63 well-defined antigenic peptides homologous with the relevant antigen sequence were identified. The contribution of each amino acid residue to the antibody binding activity of each apitope was investigated by ELISA testing of complete sets of peptide analogs containing single amino acid replacements. The data are summarized in a replaceability matrix. Some of the high frequency replaceabilities were expected, such as aspartate for glutamate, serine for threonine, etc., but unexpected relationships were also found, such as a high degree of acceptability of methionine as a replacement. Replaceability with a residue of opposite charge was rare. Glycine and tyrosine were frequently of low acceptability, except for glycine as a replacement for alanine. It was found that on average only about four to five amino acid residues in epitopes were required to determine specificity and provide binding energy. Specificity and binding energy were attributed to amino acid side chains rather than main chain atoms. Propensity factors for occurrence of amino acids in antigenic determinants were calculated. The prominence of certain hydrophobic residues as residues critical to recognition by antibody suggests that the molecular surface of an antigen in its combined from with antibody is altered from that occurring in the absence of antibody. Thus, antigenicity is not a static surface phenomenon but depends on the ability of the antigen to undergo rearrangement, supporting the induced fit concept.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 0952-3499
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The Insulin receptor is an integral transmembrane glycoprotein comprised of two α-(∼ 135kDa) and two β-(∼ 95kDa) subunits, which is synthesized as a single polypeptide chain precursor (αβ). The primary sequence of the human insulin receptor (hIR) protein, deduced from the nucleotide sequence of cloned human placental mRNAs, predicts two large domains (929 and 403 residues) on either side of a single membrane spanning domain (23 residues); each of these major domains has a distinct function (insulin binding and protein/tyrosine kinase activity, respectively). To experimentally test this deduced topology, and to explore the potential for independent domain function by the hIR extracellular domain, we have constructed an expression plasmid encoding an hIR deletion mutant which is truncated 8 residues from the beginning of the predicted transmembrane domain (i.e., 921 residues). This domain of the hIR is in fact processed into α-and truncated β-subunits and secreted with high efficiency from transfected CHO cell lines which express this mutant hIR, and the protein accumulates as an (αβ)2 dimer in the medium. This molecule is recognized by a battery of 13 monoclonal antibodies to epitopes on the IR extracellular domain, four of which block insulin binding and two of which require the native conformation of the IR for recognition. Further, this domain, binds insulin with an apparent dissociation constant comparable to that of the wild-type hIR. However, the secreted dimmer displays a linear Scatchard plot, while that of the wild-type membrane-associated hIR curvilinear. These results, together with previous results with a soluble derivative of the hIR cytoplasmic protein/tyrosine kinase domain, are consistent with the deduced topology. The IR is indeed comprised of two large soluble domains connected by a single membrane spanning domain, which on the one hand act concert upon ligand binding (insulin-dependent activation of the protein/tyrosine kinase) to initiate the insulin response in cells, but on the other hand are each capable of autonomous function (ligand binding and protein/tyrosine kinase activity, respectively).
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    ISSN: 0952-3499
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 0952-3499
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 0952-3499
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The synthesis of an 11 membered ring bis-lactam, a system which is designed as a conformationally restricted mimetic of type I and II β-turns is described. Computer assisted molecular modeling was used to compare the predicted low energy conformers of the turn mimetic with idealized type I and type II turn structures. Initial computation analysis indicates that the basic ring structure will provide an excellent foundation for the development of variety of β-turn mimetics.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 0952-3499
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Streptococcal protein G is an IgG-binding receptor with a molecular weight of 63 kDa as predicted from the sequence of the corresponding gene. Here we show that a truncated recombinant protein of 23 kDa still has IgG-binding capacity and also interacts specifically with human serum albumin (HSA). This demonstrates that protein G is a bifunctional receptor. To investigate the structures needed for IgG- and albumin-binding, different parts of the receptor molecule were produced in E. coli using a coupled expression/secretion system. Affinity Chromatography, using IgG or HSA immobilized on Sepharose, Showed that the two binding activities are structurally separated. From these experiments, it was concluded that a region of 64 amino acid residues is sufficient for albumin-binding. The structure of this part of the proteins suggests either a divalent or a trivalent binding capacity. The specific interaction to albumin was used to purify a heterologous protein by affinity chromatography to yield a pure fusion protein in a one-step procedure. The implication of this novel affinity system as a tool to facilitate protein immobilization and purification is discussed.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 0952-3499
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Most antigens recognized by T cells require unfolding or partial degradation (processing) followed by association with Major Histocompatibility Complex (MHC) molecules. We examined the processing requirements for the presentation of antigen to two T cell hybridomas which recognize the α-helical synthetic polypeptide antigen Poly 18, Poly [EYK(EYA)5], in association with I-Ad. Hybridoma A.1.1 responds to EYK(EYA)4 as the minimum antigenic sequence while hybridoma B.1.1 recognizes (EYA)5 sequence. It was found that these hybridomas responded to Poly 18 and to minimum peptide sequences presented by glutaraldehyde and chloroquine treated antigen presenting cells (APC), suggesting that antigen processing is not a requirement for the activation of these cells. The reactivity pattern of hybridoma B.1.1 in the presence of glutaraldehyde fixed APC revealed that antigens containing lysine were presented with much less efficiency than antigens without lysine, suggesting an interaction of these residues with the antigen presenting cell surface. We discuss the possibility that alanine residues in the α-helical Poly 18 form a hydrophobic ridge which may be required for appropriate interaction between antigen, the T cell receptor, and MHC molecules.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
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