Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Peptide Library”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,171 records · Page 65Linked to original sources
PubMed2000

The molecular basis of FHA domain:phosphopeptide binding specificity and implications for phospho-dependent signaling mechanisms.

Forkhead-associated (FHA) domains are a class of ubiquitous signaling modules that appear to function through interactions with phosphorylated target molecules. We have used oriented peptide library screening to determine the optimal phosphopeptide binding motifs recognized by several FHA domains, including those within a number of DNA damage checkpoint kinases, and determined the X-ray structure of Rad53p-FHA1, in complex with a phospho-threonine peptide, at 1.6 A resolution. The structure reveals a striking similarity to the MH2 domains of Smad tumor suppressor proteins and reveals a mode of peptide binding that differs from SH2, 14-3-3, or PTB domain complexes. These results have important implications for DNA damage signaling and CHK2-dependent tumor suppression, and they indicate that FHA domains play important and unsuspected roles in S/T kinase signaling mechanisms in prokaryotes and eukaryotes.

14-3-3 Proteins↗
PubMed1998

Specific interactions between the syntrophin PDZ domain and voltage-gated sodium channels.

Syntrophins are modular proteins belonging to the dystrophin associated glycoprotein complex and are thought to be involved in the regulation of the muscular system. Screening of peptide libraries revealed selectivity of the synotrophin PDZ domain toward the motif R/K/Q-E-S/T-X-V-COO- found to be highly conserved in the alpha-subunit C-terminus of vertebrate voltage gated sodium channels (VGSCs). The solution structure of the domain in complex with the peptide G-V-K-E-S-L-V shows specific interactions between the conserved residues in the peptide and syntrophin-characteristic residues in the domain. We propose that syntrophins localize VGSCs to the dystrophin network through its PDZ domain.

Amino Acid Sequence↗
PubMed2005

Rational combinatorial design of pore-forming beta-sheet peptides.

Exogenous polypeptides that self-assemble on biological membranes into pores are abundant and structurally diverse, functioning as transporters, toxins, ion channels, and antibiotics. A means for designing novel pore-forming sequences would unlock new opportunities for the development and engineering of protein function in membranes. Toward this goal, we designed a 9,604-member rational combinatorial peptide library based on the structural principles of known membrane-spanning beta-sheets. When the library was screened under stringent conditions for sequences with pore-forming activity, a single active motif was found, which is characterized by aromatic residues at the lipid-exposed interfacial positions and basic residues in the pore-lining portion of the sequence. Peptides with this motif assembled on bilayer membranes into beta-sheets and formed transient peptide/lipid pores of approximately 1-nm diameter. The mechanism of action is very similar to that of natural, pore-forming peptides. These methods provide a powerful means for selecting and engineering novel pore-forming sequences and will open prospects for designing peptide antibiotics, biosensors, and new membrane protein structures.

Amino Acid Sequence↗
PubMed2002

Mapping the C terminal epitope of the Alzheimer's disease specific antibody MN423.

The mapping of monoclonal antibody epitopes is now predominantly carried out using molecular diversity techniques, phage display in particular. However, until very recently, phage display methods have been inappropriate for the analysis of epitopes that require a free carboxy terminus. Here we describe the use of two different techniques to analyze the known C terminal epitope specificity of MN423, a monoclonal antibody specifically staining truncated tau in Alzheimer's brain. Using a lambda phage based C-terminal random peptide library, and an intracellular expression library based on truncated tau, we show that this antibody has an absolute requirement for a glycine at position -3 with respect to the C terminus. Both methods give similar results, and identify other important residues in the binding site. However, affinity analysis of synthetic peptides revealed that the affinity of the antibody for identified tripeptides was far lower than the pentapeptide sequence in the native target, and that this in turn was considerably below the affinity for the native target itself. This suggests that molecular diversity methods may define minimum, but not necessarily complete epitopes. The methods described here have a general application to the analysis of antibody epitopes suspected to be found at the C terminus.

Aged↗
PubMed2001

Peptides that mimic glycosaminoglycans: high-affinity ligands for a hyaluronan binding domain.

BACKGROUND: Hyaluronan (HA) is a non-sulfated glycosaminoglycan (GAG) that promotes motility, adhesion, and proliferation in mammalian cells, as mediated by cell-surface HA receptors. We sought to identify non-carbohydrate ligands that would bind to and activate cell-surface HA receptors. Such analogs could have important therapeutic uses in the treatment of cancer, wound healing, and arthritis, since such ligands would be resistant to degradation by hyaluronidase (HAse). RESULTS: Peptide ligands that bind specifically to the recombinant HA binding domain (BD) of the receptor for hyaluronan-mediated motility (RHAMM) were obtained by screening two peptide libraries: (i) random 8-mers and (ii) biased 8-mers with alternating acidic side chains, i.e. XZXZXZXZ (X=all-L-amino acids except Cys, Lys, or Arg; Z=D-Asp, L-Asp, D-Glu, or L-Glu). Selectivity of the peptide ligands for the HABD was established by (i) detection of binding of biotin- or fluorescein-labeled peptides to immobilized proteins and (ii) fluorescence polarization of FITC-labeled peptides with the HABD in solution. HA competitively displaced binding of peptides to the HABD, while other GAGs were less effective competitors. The stereochemistry of four biased octapeptides was established by synthesis of the 16 stereoisomers of each peptide. Binding assays demonstrated a strong preference for alternating D and L configurations for the acidic residues, consistent with the calculated orientation of glucuronic acid moieties of HA. CONCLUSIONS: Two classes of HAse-resistant peptide mimetics of HA were identified with high affinity, HA-compatible binding to the RHAMM HABD. This demonstrated that non-HA ligands specific to a given HA binding protein could be engineered, permitting receptor-specific targeting.

Amino Acid Sequence↗
PubMed2000

Assessment of protein-tyrosine phosphatase 1B substrate specificity using "inverse alanine scanning".

An "inverse alanine scanning" peptide library approach has been developed to assess the substrate specificity of protein-tyrosine phosphatases (PTPases). In this method each Ala moiety in the parent peptide, Ac-AAAApYAAAA-NH(2), is separately and sequentially replaced by the 19 non-Ala amino acids to generate a library of 153 well defined peptides. The relatively small number of peptides allows the acquisition of explicit kinetic data for all library members, thereby furnishing information about the contribution of individual amino acids with respect to substrate properties. The approach was applied to protein-tyrosine phosphatase 1B (PTP1B) as a first example, and the highly potent peptide substrate Ac-ELEFpYMDYE-NH(2) (k(cat)/K(m) 2.2 +/- 0.05 x 10(7) M(-1) s(-1)) has been identified. More importantly, several heretofore unknown features of the substrate specificity of PTP1B were revealed. This includes the ability of PTP1B to accommodate acidic, aromatic, and hydrophobic residues at the -1 position, a strong nonpreference for Lys and Arg residues in any position, and the first evidence that residues well beyond the +1 position contribute to substrate efficacy.

Alanine↗
PubMed2006

Relating TCR-peptide-MHC affinity to immunogenicity for the design of tumor vaccines.

One approach to enhancing the T cell response to tumors is vaccination with mimotopes, mimics of tumor epitopes. While mimotopes can stimulate proliferation of T cells that recognize tumor-associated antigens (TAAs), this expansion does not always correlate with control of tumor growth. We hypothesized that vaccination with mimotopes of optimal affinity in this interaction will improve antitumor immunity. Using a combinatorial peptide library and a cytotoxic T lymphocyte clone that recognizes a TAA, we identified a panel of mimotopes that, when complexed with MHC, bound the TAA-specific TCR with a range of affinities. As expected, in vitro assays showed that the affinity of the TCR-peptide-MHC (TCR-pMHC) interaction correlated with activity of the T cell clone. However, only vaccination with mimotopes in the intermediate-affinity range elicited functional T cells and provided protection against tumor growth in vivo. Vaccination with mimotopes with the highest-affinity TCR-pMHC interactions elicited TAA-specific T cells to the tumor, but did not control tumor growth at any of the peptide concentrations tested. Further analysis of these T cells showed functional defects in response to the TAA. Thus, stimulation of an antitumor response by mimotopes may be optimal with peptides that increase but do not maximize the affinity of the TCR-pMHC interaction.

Animals↗
PubMed2000

Structure of the FHA1 domain of yeast Rad53 and identification of binding sites for both FHA1 and its target protein Rad9.

Forkhead-associated (FHA) domains have been shown to recognize both pThr and pTyr-peptides. The solution structures of the FHA2 domain of Rad53 from Saccharomyces cerevisiae, and its complex with a pTyr peptide, have been reported recently. We now report the solution structure of the other FHA domain of Rad53, FHA1 (residues 14-164), and identification of binding sites of FHA1 and its target protein Rad9. The FHA1 structure consists of 11 beta-strands, which form two large twisted anti-parallel beta-sheets folding into a beta-sandwich. Three short alpha-helices were also identified. The beta-strands are linked by several loops and turns. These structural features of free FHA1 are similar to those of free FHA2, but there are significant differences in the loops. Screening of a peptide library [XXX(pT)XXX] against FHA1 revealed an absolute requirement for Asp at the +3 position and a preference for Ala at the +2 position. These two criteria are met by a pThr motif (192)TEAD(195) in Rad9. Surface plasmon resonance analysis showed that a pThr peptide containing this motif, (188)SLEV(pT)EADATFVQ(200) from Rad9, binds to FHA1 with a K(d) value of 0.36 microM. Other peptides containing pTXXD sequences also bound to FHA1, but less tightly (K(d)=4-70 microM). These results suggest that Thr192 of Rad9 is the likely phosphorylation site recognized by the FHA1 domain of Rad53. The tight-binding peptide was then used to identify residues of FHA1 involved in the interaction with the pThr peptide. The results are compared with the interactions between the FHA2 domain and a pTyr peptide derived from Rad9 reported previously.

Amino Acid Motifs↗
PubMed2004

The interaction of an antimicrobial decapeptide with phospholipid vesicles.

Previously, by using combinatorial peptide libraries, we have identified activity-optimized decapeptide (KSL, KKVVFKVKFK-NH(2)), which exhibited a broad spectrum of the activity against bacteria and fungi without hemolytic activity. In order to examine lipid requirements and to understand the mode of KSL action, we investigated interactions of the peptide with vesicles consisting of various lipid compositions. KSL increased the permeability of negatively charged but not zwitterionic phospholipid membranes, and the leakage was independent on the size of encapsulated molecules (calcein, 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS)/N,N'-p-xylene bis(pyridinium) bromide (DPX), and fluorescein isothiocyanate (FITC)-dextran with different molecular weight), indicating that the peptide did not form pores or channels in this leakage process. KSL ability to permeabilize vesicles with negatively charged surface was dramatically reduced upon the addition of zwitterionic phospholipid rather than cholesterol, which revealed that the surface charge of lipid membranes played a major role in the activity and selectivity of KSL. Moreover, KSL diastereomer did not increase the permeability of negatively charged vesicles, indicating that the secondary structure of KSL was also required for membrane perturbation activity. Interestingly, KSL had an ability to cause aggregation and subsequent fusion of the acidic vesicles, which seemed to be related to the biological action. Structural studies performed by circular dichroism (CD) spectroscopy indicated that in the presence of acidic vesicles, the beta sheet structure of KSL must be required for the ability to (1) induce a leakage of dye from the acidic vesicles (2) to fuse the acidic vesicles.

Anti-Bacterial Agents↗
PubMed2004

Vascular zip codes in angiogenesis and metastasis.

In vivo screening of phage-displayed peptide libraries has revealed extensive molecular differences in the blood vessels of individual normal tissues. Pathological lesions also put their signature on the vasculature; in tumours, both blood and lymphatic vessels differ from normal vessels. The changes that characterize tumour blood vessels include selective expression of certain integrins. Peptides isolated by in vivo phage display for homing to tumours have been shown to be useful in directing therapeutic agents to experimental tumours. The targeting can enhance the efficacy of the therapy while reducing side effects. Phage screening has also revealed lung-specific vascular markers that promote tumour metastasis to the lungs by mediating specific adherence of tumour cells to the lung vasculature. These phage-screening studies have revealed a previously unsuspected degree of vascular specialization and provide potentially useful guidance devices for targeted therapies.

Animals↗
PubMed2003

Development of peptide mimotopes of lipooligosaccharide from nontypeable Haemophilus influenzae as vaccine candidates.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of otitis media in children and lower respiratory tract diseases in adults. So far there is no effective vaccine against NTHi. A major surface-exposed component of NTHi, lipooligosaccharide (LOS), is a virulence factor as well as a potential protective Ag. LOS is too toxic to be administered in humans. However, detoxified LOS is a T cell-independent small molecule and is poorly immunogenic in vivo, so we converted LOS into a nontoxic T cell-dependent Ag through the use of peptides that mimic the LOS by screening a phage-display peptide library with a rabbit Ab specific for NTHi LOS. Fifty-six phage clones were found to share LOS mimicry molecules. Among them, 22 clones were subjected to DNA sequencing, and four consensus sequences were identified as NMMRFTSQPPNN, NMMNYIMDPRTH, NMMKYISPPIFL, and NMMRFTELSTPS. Three of the four synthetic peptides showed strong binding reactivity to the rabbit anti-LOS Ab and also a mouse bactericidal monoclonal anti-LOS Ab in vitro, and elicited specific serum anti-LOS Abs in rabbits (27- to 81-fold) after conjugation with keyhole limpet hemocyanin. Passive immunization with the rabbit antisera resulted in a significantly enhanced pulmonary bacterial clearance in a mouse model. The enhanced bacterial clearance was eliminated if the rabbit serum was preabsorbed with NTHi LOS. These data indicate that the peptide mimotopes of LOS that we have identified might be potential components of peptide vaccines against NTHi.

Animals↗
PubMed2005

Cytokine recognition by human interleukin 5 receptor.

The activation of interleukin 5 (IL-5) receptor is a dynamic process that depends on specific interaction of IL-5 with IL-5 receptor alpha, the formation of oligomeric receptor complexes with receptor beta, and the initiation of cytoplasmic phosphorylation events. These steps culminate in the triggering of a cellular response. Important advances have been made recently in understanding the molecular mechanisms of cytokine recognition, receptor assembly, and signal triggering. Cytokine recognition can be envisioned by relating structure to function in IL-5 and IL-5 receptor alpha. A pair of charge-complementary regions plays an essential role in the specific interaction between IL-5 receptor alpha and IL-5. Moreover, peptide library methodology has led to the discovery of IL-5 receptor alpha antagonists that mimic key elements in IL-5 receptor recognition. Because IL-5 has been implicated in the pathology of eosinophil-related inflammatory diseases, revealing the key recognition elements of IL-5, IL-5 mimetic peptides, and IL-5 receptor alpha could help drive the design of new compounds for therapeutic treatment against allergic inflammatory diseases such as asthma.

Animals↗
PubMed2000

Contribution of the different modules in the utrophin carboxy-terminal region to the formation and regulation of the DAP complex.

The carboxy-terminal region of utrophin, like the homologous proteins dystrophin, Drp2 and dystrobrevins, contains structural domains frequently involved in protein-protein interaction. These domains (WW, EF hands, ZZ and H1-H2) mediate recognition and binding to a multicomponent complex of proteins, also known as dystrophin-associated proteins (DAPs) for their association with dystrophin, the product of the gene, mutated in Duchenne muscular dystrophy. We have exploited phage display and in vitro binding assays to study the recognition specificity of the different domains of the utrophin carboxy-terminus. We found that none of the carboxy-terminal domains of utrophin, when isolated from its structural context, selects specific ligand peptides from a phage-displayed peptide library. By contrast, panning with an extended region containing the WW, EF hands, and ZZ domain defines the consensus binding motif, PPxY which is also found in beta-dystroglycan, a component of the DAP complex that interacts with utrophin in several tissues. WW-mediated binding to PPxY peptides and to beta-dystroglycan requires the presence of the EF hands and ZZ domain. When the ZZ domain is either deleted or engaged in binding to calmodulin, the utrophin beta-dystroglycan complex cannot be formed. These findings suggest a potential regulatory mechanism by means of which the attachment of utrophin to the DAP complex can be modulated by the Ca(2+)-dependent binding of calmodulin. The remaining two motifs found in the carboxy-terminus (H1-H2) mediate the formation of utrophin-dystrobrevin hybrids but do not select ligands in a repertoire of random nonapeptides.

Amino Acid Sequence↗
PubMed1999

NG2 proteoglycan-binding peptides target tumor neovasculature.

NG2 is the rat homologue of the human melanoma proteoglycan, also known as the high molecular weight melanoma-associated antigen. This developmentally regulated membrane-spanning chondroitin sulfate proteoglycan is expressed primarily by glial, muscle, and cartilage progenitor cells. Upon maturation, these cell types down-regulate NG2 expression. In adult animals, the expression of NG2 is restricted to tumor cells and angiogenic tumor vasculature, making this proteoglycan a potential target for directing therapeutic agents to relevant sites of action. To this end, we have identified specific NG2-binding peptides by screening a phage-displayed random peptide library on purified NG2. Several rounds of biopanning on NG2 resulted in the specific enrichment of two phage-displayed decapeptides, TAASGVRSMH and LTLRWVGLMS. The binding of these phages to NG2 was inhibitable both by soluble NG2 and by glutathione S-transferase (GST) fusion proteins containing the cognate peptide sequences. In addition, direct binding between GST-TAASGVRSMH and GST-LTLRWVGLMS fusion proteins and NG2 was demonstrated in solid-phase binding assays. Interestingly, these NG2-binding fusion proteins cross-inhibited each other's binding to NG2, suggesting that the two sequences bind to the same or overlapping sites on the proteoglycan. Upon injection into tumor-bearing mice, NG2-binding phages specifically homed to tumor vasculature in wild-type mice but did not localize to the tumor vasculature in NG2 knockout mice. The in vivo targeting capability of these sequences suggests that they can be used for tumor targeting.

Animals↗
PubMed1999

Actin surface structure revealed by antibody imprints: evaluation of phage-display analysis of anti-actin antibodies.

Phage-display peptide library analysis of an anti-F actin polyclonal antibody identified 12 amino acid residues of actin that appear, in its X-ray crystal structure, to be grouped together in a surface accessible conformational epitope. Phage epitope mapping was carried out by isolating immune complexes containing members of the J404 nonapeptide phage-display library formed in diluted antiserum and isolated on a protein A affinity matrix. Immunoreactive clones were grown as plaques, replica plated onto nitrocellulose, and labeled with anti-actin immune serum. One hundred and forty-four positively staining clones identified in this way were sequenced. Of these, 54 displayed peptides with sequence similarities. When the most abundantly selected sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on Western blots and inhibited the binding of the antibody to immobilized F-actin by 60%. A scrambled version of this sequence WQDK WLQTQ, when coupled to ovalbumin, was not recognized by the antiserum and minimally inhibited binding of antiserum to immobilized F-actin by 10%. KQTWQQLWD contained four residues that corresponded, in frame, to a highly conserved six residue region of the chicken beta-actin sequence 351TFQQMW356 (identical residues are shown in bold). Examination of the rabbit skeletal muscle X-ray crystal structure suggested that within a 15 A radius of W356, nine additional residues were arranged on the actin surface in such a way that they could be mimicked by several of the selected phage sequences with root-mean-square deviation fits of 2.1-2.5 A. We conclude that phage-display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.

Actins↗
PubMed2006

The membrane-active regions of the hepatitis C virus E1 and E2 envelope glycoproteins.

We have identified the membrane-active regions of the full sequences of the HCV E1 and E2 envelope glycoproteins by performing an exhaustive study of membrane leakage, hemifusion, and fusion induced by 18-mer peptide libraries on model membranes having different phospholipid compositions. The data and their comparison have led us to identify different E1 and E2 membrane-active segments which might be implicated in viral membrane fusion, membrane interaction, and/or protein-protein binding. Moreover, it has permitted us to suggest that the fusion peptide might be located in the E1 glycoprotein and, more specifically, the segment comprised by amino acid residues 265-296. The identification of these membrane-active segments from the E1 and E2 envelope glycoproteins, as well as their membranotropic propensity, supports their direct role in HCV-mediated membrane fusion, sustains the notion that different segments provide the driving force for the merging of the viral and target cell membranes, and defines those segments as attractive targets for further development of new antiviral compounds.

Binding Sites↗
PubMed2006

Peripheral region for core cross-beta plays important role in amyloidogenicity.

The role of the peripheral sequence neighboring the core cross-beta region was investigated using a peptide library constructed with all possible combinations of Lys, Glu, Ser, and Leu at three residue positions (X1-X3) forming the N-terminal region linked to the amyloid core sequence of the barnase-derived segment (A4-K22). By means of CD spectra and thioflavin T binding assay for 64 peptides, not only the composition but also the sequence in the peripheral region were found to be responsible for amyloid formation. The preferences of amino acid residues in the peripheral region of the amyloid-forming peptides were in the order of Leu approximately SerGlu>>Lys. A balance of positive and negative charges was found to be essential for amyloid formation, suggesting that the electrostatic interaction at the surface of the amyloid fibrils is relevant to their stability. On the basis of the maximum fluorescence wavelength of fibril-bound thioflavin T, the highly amyloidogenic peptides were classified into two classes, which exhibited the sequence preferences of (Leu, Ser/Glu, and Leu) and (Glu, Leu, and Ser) for the peripheral sequence (X1, X2, and X3). The former class can be rationally assigned to the structural model with deep grooves along the fibril axis. Thus, the peripheral sequence regulates the manner of molecular packing in the fibrils as well as the amyloidogenicity. In addition, the chains of the peripheral sequence are most likely to form thioflavin T binding sites.

Amino Acids↗
PubMed1997

Identification of phosphopeptide ligands for the Src-homology 2 (SH2) domain of Grb2 by phage display.

We report here on the identification of phosphopetide ligands which interact with the Src-homology 2 (SH2) domain of the adapter protein Grb2 by screening a random peptide library established on phage. Phage were phosphorylated in vitro at an invariant tyrosine residue by a mixture of phosphotyrosine kinases c-Src, Blk and Syk. Selection of binding motifs was carried out by interaction of the library with the recombinant SH2 domain of Grb2 expressed as a glutathione S-transferase (GST) fusion protein. Several subsequent cycles of selection led to the enrichment of phage which bound to the GST-Grb2 SH2 domain only when previously phosphorylated. Sequence analysis revealed that all of the selected phage displayed peptides with the consensus motif Y*M/ENW (Y* denotes phosphotyrosine). One of these peptides, bearing the Y*ENW motif, bound the Grb2 SH2 domain with a threefold higher affinity than the peptide motif Y*VNV derived from the natural ligand Shc. Thus, phage display can be employed to rapidly identify high affinity ligands to SH2 domains.

Adaptor Proteins, Signal Transducing↗