Home About us Contact
|
Home About us Contact | ||
|
|
||
Peptide Binding (peptide + binding)
Terms modified by Peptide Binding Selected AbstractsRationally designed mutations convert complexes of human recombinant T cell receptor ligands into monomers that retain biological activityJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 1 2005Jianya Y Huan Abstract Single-chain human recombinant T cell receptor ligands derived from the peptide binding/TCR recognition domain of human HLA-DR2b (DRA*0101/DRB1*1501) produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides have been described previously. While molecules with the native sequence retained biological activity, they formed higher order aggregates in solution. In this study, we used site-directed mutagenesis to modify the ,-sheet platform of the DR2-derived RTLs, obtaining two variants that were monomeric in solution by replacing hydrophobic residues with polar (serine) or charged (aspartic acid) residues. Size exclusion chromatography and dynamic light scattering demonstrated that the modified RTLs were monomeric in solution, and structural characterization using circular dichroism demonstrated the highly ordered secondary structure of the RTLs. Peptide binding to the ,empty' RTLs was quantified using biotinylated peptides, and functional studies showed that the modified RTLs containing covalently tethered peptides were able to inhibit antigen-specific T cell proliferation in vitro, as well as suppress experimental autoimmune encephalomyelitis in vivo. These studies demonstrated that RTLs encoding the Ag-binding/TCR recognition domain of MHC class II molecules are innately very robust structures, capable of retaining potent biological activity separate from the Ig-fold domains of the progenitor class II structure, with prevention of aggregation accomplished by modification of an exposed surface that was buried in the progenitor structure. Copyright © 2004 Society of Chemical Industry [source] Pharmacodynamics and pharmacokinetics of YM128, a GPIIb/IIIa antagonist prodrugDRUG DEVELOPMENT RESEARCH, Issue 3 2002Ken-ichi Suzuki Abstract We examined the biochemical properties of YM-57029 ({4-[4-(4-Carbamimidoylphenyl)-3-oxopiperazin-1-yl]piperidino}acetic acid monohydrochloride trihydrate) and the pharmacodynamics and pharmacokinetics of its prodrug, YM128 (Ethyl (Z)-(4-{4-[4-(N2 -hydroxycarbamimidoyl)phenyl]-3-oxopiperazin-1-yl}piperidino)acetate), an orally-active glycoprotein IIb/IIIa (GPIIb/IIIa) antagonist. YM-57029 strongly inhibited aggregation of human platelets induced by various agonists, with IC50 values ranging from 3.6 to 51 nM. YM-57029 specifically inhibited fibrinogen binding to purified GPIIb/IIIa about 1,000-fold more potently than Arg-Gly-Asp-Ser (RGDS). Moreover, YM-57029 effectively inhibited an Arg-Gly-Asp (RGD) peptide binding to platelets, suggesting that YM-57029 competed with the RGD sequence of ligand. YM-57029 or YM128 dose-dependently inhibited ex vivo platelet aggregation after iv bolus injection or oral administration to beagle dogs and cynomolgus monkeys. However, YM128 exerted more potent and prolonged inhibitory effects on platelet aggregation than YM-57029 after oral administration to cynomolgus monkeys. Furthermore, YM-57029 prolonged template bleeding time at a dose that inhibited ex vivo platelet aggregation during cumulative iv infusion to cynomolgus monkeys. Metabolic and pharmacokinetic studies showed that YM128 effectively converted into YM-57029 in liver microsomes from humans as well as dogs and monkeys, and that bioavailabilities of YM128 in dogs and monkeys were 32.3 and 22.2%, respectively. These results suggest that YM128, a prodrug of YM-57029, may be a valuable GPIIb/IIIa antagonist with good bioavailability in humans. Drug Dev. Res. 55:149,161, 2002. © 2002 Wiley-Liss, Inc. [source] Major histocompatibility complex class I binding predictions as a tool in epitope discoveryIMMUNOLOGY, Issue 3 2010Claus Lundegaard Summary Over the last decade, in silico models of the major histocompatibility complex (MHC) class I pathway have developed significantly. Before, peptide binding could only be reliably modelled for a few major human or mouse histocompatibility molecules; now, high-accuracy predictions are available for any human leucocyte antigen (HLA) -A or -B molecule with known protein sequence. Furthermore, peptide binding to MHC molecules from several non-human primates, mouse strains and other mammals can now be predicted. In this review, a number of different prediction methods are briefly explained, highlighting the most useful and historically important. Selected case stories, where these ,reverse immunology' systems have been used in actual epitope discovery, are briefly reviewed. We conclude that this new generation of epitope discovery systems has become a highly efficient tool for epitope discovery, and recommend that the less accurate prediction systems of the past be abandoned, as these are obsolete. [source] Sequence flexibility of the immunodominant HLA A*0201 restricted ppUL83 CD8 T-cell epitope of human cytomegalovirusJOURNAL OF MEDICAL VIROLOGY, Issue 1 2010Jakub Kopycinski Abstract The cytomegalovirus ppUL83 protein contains an immunodominant A*0201 restricted epitope between residues 495 and 503. We investigated the tolerance of this epitope to sequence variation in the context of peptide binding to HLA A*0201 and the ability to induce an Interferon gamma (IFN,) response through engagement with the T-cell receptor (TCR). The majority of mutations investigated resulted in a decrease in the production of IFN, indicating that if such variants occurred in vivo they would not be recognized by CD8 T-cell clones specific for the wild-type epitope. The mechanistic basis for the majority of the mutant peptides was their failure to bind and stabilize class I HLA cell surface expression. However, one peptide with a mutation at the P5 position (methionine to cysteine) resulted in a significant enhanced binding to HLA A*0201 and also an increase in cell surface expression over the wild-type peptide but was unable to engage with the CD8 TCR and trigger IFN, production. This peptide acted as a competitive inhibitor of the wild-type peptide but could not fully inhibit IFN, production by the latter. We subsequently investigated whether mutations of the HLA A*0201 epitope were evident in immunocompromized patients experiencing either rapid exponential or persistent cytomegalovirus replication. J. Med. Virol. 82:94,103, 2010. © 2009 Wiley-Liss, Inc. [source] Characterization of prostate-specific antigen binding peptides selected by phage display technologyJOURNAL OF MOLECULAR RECOGNITION, Issue 1 2006Catherine Ferrieu-Weisbuch Abstract Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer. Free PSA has been shown to be more extensively cleaved in sera from benign prostatic hyperplasia patients than in sera from prostate cancer patients. Moreover, the presence of enzymatically activatable PSA was characterized previously in sera from patients with prostate cancer by the use of the specific anti-free PSA monoclonal antibody (mAb) 5D3D11. As an attempt to obtain ligands for the specific recognition of different PSA forms including active PSA, phage-displayed linear and cyclic peptide libraries were screened with PSA coated directly into microplate wells or presented by two different anti-total PSA mAbs. Four different phage clones were selected for their ability to recognize PSA and the inserted peptides were produced as synthetic peptides. These peptides were found to capture and to detect specifically free PSA, even in complex biological media such as sera or tumour cell culture supernatants. Alanine scanning of peptide sequences showed the involvement of aromatic and hydrophobic residues in the interaction of the peptides with PSA whereas Spotscan analysis of overlapping peptides covering the PSA sequence identified a peptide binding to the kallikrein loop at residues 82,87, suggesting that the peptides could recognize a non-clipped form of PSA. Moreover, the PSA-specific peptides enhance the enzymatic activity of PSA immobilized into microplate wells whereas the capture of PSA by the peptides inhibited totally its enzymatic activity while the peptide binding to PSA had no effect in solution. These PSA-specific peptides could be potential tools for the recognition of PSA forms more specifically associated to prostate cancer. Copyright © 2005 John Wiley & Sons, Ltd. [source] Applications of model ,-hairpin peptidesJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 12 2004Carol E. Stotz Abstract In recent years, ,-hairpin peptides have been studied in great detail. Much of the focus has been on the thermodynamic stability of ,-hairpin structure. Structural measurements have been conducted with nuclear magnetic resonance, with additional information obtained from circular dichroism, Fourier transform infrared, and molecular dynamic simulation studies. Point mutations, both in the ,-strands and in the turn region, have systematically explored the role of turn sequence, side-chain,side-chain interactions, intramolecular hydrogen bonding, and ,-strand length on ,-hairpin peptide conformational stability. In addition to studying the elements of structural stability independently, the cooperative nature of the individual components to combine to form the overall structure has also been investigated. Because the ,-hairpin peptides often spontaneously form their conformation, they have begun to serve as models for studying peptide binding and therapeutic agents. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:2881,2894, 2004 [source] Side chain substitution benchmark for peptide/MHC interactionPROTEIN SCIENCE, Issue 6 2008Bernhard Knapp Abstract The prediction of T-cell epitopes is an essential part in virtual immunology. Apart from sequence-based techniques, which achieve good results but fail to give insight into the binding behavior of a certain peptide binding to a major histocompatibility complex, structure-based approaches are another important technique. An essential step is the correct placement of the side chains for a given peptide in cases where no experimental data for the structure are available. To our knowledge, no benchmark for side chain substitution in the area of HLA has been reported in the literature. Here, we present a comparison of five different tools (SCWRL, SCATD, SPDBV, SCit, IRECS) applicable for side chain substitution. Each tool is tested on 29 different HLA-A2 structures with experimentally known side chain positions. Parts of the benchmark are correctness, reliability, runtime, and usability. For validation, the root mean square deviations between X-ray structures and predicted structures are used. All tools show different strengths and weaknesses. [source] The identification of conserved interactions within the SH3 domain by alignment of sequences and structuresPROTEIN SCIENCE, Issue 11 2000Stefan M. Larson Abstract The SH3 domain, comprised of approximately 60 residues, is found within a wide variety of proteins, and is a mediator of protein,protein interactions. Due to the large number of SH3 domain sequences and structures in the databases, this domain provides one of the best available systems for the examination of sequence and structural conservation within a protein family. In this study, a large and diverse alignment of SH3 domain sequences was constructed, and the pattern of conservation within this alignment was compared to conserved structural features, as deduced from analysis of eighteen different SH3 domain structures. Seventeen SH3 domain structures solved in the presence of bound peptide were also examined to identify positions that are consistently most important in mediating the peptide-binding function of this domain. Although residues at the two most conserved positions in the alignment are directly involved in peptide binding, residues at most other conserved positions play structural roles, such as stabilizing turns or comprising the hydrophobic core. Surprisingly, several highly conserved side-chain to main-chain hydrogen bonds were observed in the functionally crucial RT-Src loop between residues with little direct involvement in peptide binding. These hydrogen bonds may be important for maintaining this region in the precise conformation necessary for specific peptide recognition. In addition, a previously unrecognized yet highly conserved ,-bulge was identified in the second ,-strand of the domain, which appears to provide a necessary kink in this strand, allowing it to hydrogen bond to both sheets comprising the fold. [source] Flexibility of the MHC class II peptide binding cleft in the bound, partially filled, and empty states: A molecular dynamics simulation studyBIOPOLYMERS, Issue 1 2009Rakina Yaneva Abstract Major histocompatibility (MHC) Class II cell surface proteins present antigenic peptides to the immune system. Class II structures in complex with peptides but not in the absence of peptide are known. Comparative molecular dynamics (MD) simulations of a Class II protein (HLA-DR3) with and without CLIP (invariant chain-associated protein) peptide were performed starting from the CLIP-bound crystal structure. Depending on the protonation of acidic residues in the P6 peptide-binding pocket the simulations stayed overall close to the start structure. The simulations without CLIP showed larger conformational fluctuations especially of ,-helices flanking the binding cleft. Largest fluctuations without CLIP were observed in a helical segment near the peptide C-terminus binding region matching a segment recognized by antibodies specific for empty Class II proteins. Simulations on a Val86Tyr mutation that fills the peptide N-terminus binding P1 pocket or of a complex with a CLIP fragment (dipeptide) bound to P1 showed an unexpected long range effect. In both simulations the mobility not only of P1 but also of the entire binding cleft was reduced compared to simulations without CLIP. It correlates with the experimental finding that the CLIP fragment binding to P1 is sufficient to prevent antibody recognition specific for the empty form at a site distant from P1. The results suggest a mechanism how a local binding event of small peptides or of an exchange factor near P1 may promote peptide binding and exchange through a long range stabilization of the whole binding cleft in a receptive (near bound) conformation. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 14,27, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] DNA and RNA-Controlled Switching of Protein Kinase ActivityCHEMBIOCHEM, Issue 4 2009Lars Röglin Dr. Abstract Constrained: The readily programmable nucleic acid mediated recognition is used to constrain a phosphopeptide that was flanked by PNA segments. RNA-based switching allows control over the activity of target enzymes such as the protein kinase Src. It might thus be feasible to transduce changes of the concentration of selected RNA molecules to changes of the activity of signal transduction proteins. Protein switches use the binding energy gained upon recognition of ligands to modulate the conformation and binding properties of protein segments. We explored whether the programmable nucleic acid mediated recognition might be used to design or mimic constraints that limit the conformational freedom of peptide segments. The aim was to design nucleic acid,peptide conjugates in which the peptide portion of the conjugate would change the affinity for a protein target upon hybridization. This approach was used to control the affinity of a PNA,phosphopeptide conjugate for the signal transduction protein Src kinase, which binds the cognate phosphopeptides in a linear conformation. Peptide,nucleic acid arms were attached to known peptide binders. The chimeric molecules were studied in three modes: 1) as single strands, 2) constrained by intermolecular hybridization (duplex formation) and 3) constrained by intramolecular hybridization (hairpin formation). Of note, duplexes that were designed to accommodate bulged peptide structures (for example, in hairpins or bulges) had lower binding affinities than duplexes in which the peptide was allowed to adopt a more relaxed conformation. Greater than 90-fold differences in binding affinities were observed. It was, thus, feasible to make use of DNA hybridization to reversibly switch from no to almost complete inhibition of Src-SH2,peptide binding, and vice versa. A series of DNA and PNA-based hybridization experiments revealed the importance of charges and conformational effects. Nucleic acid mediated switching was extended to the use of RNA; this enabled a regulation of the enzymatic activity of the Src kinase. The proof-of-principle results demonstrate for the first time that PNA,peptide chimeras can transduce changes of the concentration of a given RNA molecule to changes of the activity of a signal transduction enzyme. [source] | |||||||||||||