Discovery of a polystyrene binding peptide isolated from phage display library and its application in peptide immobilization.

PubMed

Qiang, Xu; Sun, Keyong; Xing, Lijun; Xu, Yifeng; Wang, Hong; Zhou, Zhengpin; Zhang, Juan; Zhang, Fang; Caliskan, Bilgen; Wang, Min; Qiu, Zheng

2017-06-01

Phage peptide display is a powerful technique for discovery of various target-specific ligands. However, target-unrelated peptides can often be obtained and cause ambiguous results. Peptide PB-TUP has been isolated repeatedly in our laboratory on different targets and we conducted a research on PB-TUP phage to investigate their binding properties and rate of propagation. ELISA and phage recovery assay demonstrated that PB-TUP phage had a significant superior affinity to polystyrene solid surface compared with control phage clones. In this study, some incidental bindings are excluded like blocking agents and non-specific binding of secondary antibodies. Propagation rate assays of the selected phage clones showed that the growth rate of PB-TUP phage was not superior to the control phages. Furthermore, the binding of PB-TUB to polystyrene was concentration dependent and varied with solution pH. Molecular modeling revealed that stable structures of α-helix and β-turn may contribute to the binding of PB-TUP to polystyrene plate. The PB-TUP sequence was fused to the N-terminus of peptide P2 and the fusion peptide significantly increased the binding affinity to polystyrene. The fusion peptide also enhanced the cell adhesion ability of peptide P2 with human umbilical vein endothelial cell (HUVEC). The addition of the polystyrene binding peptide provided a convenient method for peptide immobilization.

Elucidation of the binding preferences of peptide recognition modules: SH3 and PDZ domains.

PubMed

Teyra, Joan; Sidhu, Sachdev S; Kim, Philip M

2012-08-14

Peptide-binding domains play a critical role in regulation of cellular processes by mediating protein interactions involved in signalling. In recent years, the development of large-scale technologies has enabled exhaustive studies on the peptide recognition preferences for a number of peptide-binding domain families. These efforts have provided significant insights into the binding specificities of these modular domains. Many research groups have taken advantage of this unprecedented volume of specificity data and have developed a variety of new algorithms for the prediction of binding specificities of peptide-binding domains and for the prediction of their natural binding targets. This knowledge has also been applied to the design of synthetic peptide-binding domains in order to rewire protein-protein interaction networks. Here, we describe how these experimental technologies have impacted on our understanding of peptide-binding domain specificities and on the elucidation of their natural ligands. We discuss SH3 and PDZ domains as well characterized examples, and we explore the feasibility of expanding high-throughput experiments to other peptide-binding domains. Copyright © 2012. Published by Elsevier B.V.

Biopanning and characterization of peptides with Fe3O4 nanoparticles-binding capability via phage display random peptide library technique.

PubMed

You, Fei; Yin, Guangfu; Pu, Ximing; Li, Yucan; Hu, Yang; Huang, Zhongbin; Liao, Xiaoming; Yao, Yadong; Chen, Xianchun

2016-05-01

Functionalization of inorganic nanoparticles (NPs) play an important role in biomedical applications. A proper functionalization of NPs can improve biocompatibility, avoid a loss of bioactivity, and further endow NPs with unique performances. Modification with vairous specific binding biomolecules from random biological libraries has been explored. In this work, two 7-mer peptides with sequences of HYIDFRW and TVNFKLY were selected from a phage display random peptide library by using ferromagnetic NPs as targets, and were verified to display strong binding affinity to Fe3O4 NPs. Fourier transform infrared spectrometry, fluorescence microscopy, thermal analysis and X-ray photoelectron spectroscopy confirmed the presence of peptides on the surface of Fe3O4 NPs. Sequence analyses revealed that the probable binding mechanism between the peptide and Fe3O4 NPs might be driven by Pearson hard acid-hard base specific interaction and hydrogen bonds, accompanied with hydrophilic interactions and non-specific electrostatic attractions. The cell viability assay indicated a good cytocompatibility of peptide-bound Fe3O4 NPs. Furthermore, TVNFKLY peptide and an ovarian tumor cell A2780 specific binding peptide (QQTNWSL) were conjugated to afford a liner 14-mer peptide (QQTNWSLTVNFKLY). The binding and targeting studies showed that 14-mer peptide was able to retain both the strong binding ability to Fe3O4 NPs and the specific binding ability to A2780 cells. The results suggested that the Fe3O4-binding peptides would be of great potential in the functionalization of Fe3O4 NPs for the tumor-targeted drug delivery and magnetic hyperthermia. Copyright © 2016 Elsevier B.V. All rights reserved.

The structural basis for function in diamond-like carbon binding peptides.

PubMed

Gabryelczyk, Bartosz; Szilvay, Géza R; Linder, Markus B

2014-07-29

The molecular structural basis for the function of specific peptides that bind to diamond-like carbon (DLC) surfaces was investigated. For this, a competition assay that provided a robust way of comparing relative affinities of peptide variants was set up. Point mutations of specific residues resulted in significant effects, but it was shown that the chemical composition of the peptide was not sufficient to explain peptide affinity. More significantly, rearrangements in the sequence indicated that the binding is a complex recognition event that is dependent on the overall structure of the peptide. The work demonstrates the unique properties of peptides for creating functionality at interfaces via noncovalent binding for potential applications in, for example, nanomaterials, biomedical materials, and sensors.

Viral peptides-MHC interaction: Binding probability and distance from human peptides.

PubMed

Santoni, Daniele

2018-05-23

Identification of peptides binding to MHC class I complex can play a crucial role in retrieving potential targets able to trigger an immune response. Affinity binding of viral peptides can be estimated through effective computational methods that in the most of cases are based on machine learning approach. Achieving a better insight into peptide features that impact on the affinity binding rate is a challenging issue. In the present work we focused on 9-mer peptides of Human immunodeficiency virus type 1 and Human herpes simplex virus 1, studying their binding to MHC class I. Viral 9-mers were partitioned into different classes, where each class is characterized by how far (in terms of mutation steps) the peptides belonging to that class are from human 9-mers. Viral 9-mers were partitioned in different classes, based on the number of mutation steps they are far from human 9-mers. We showed that the overall binding probability significantly differs among classes, and it typically increases as the distance, computed in terms of number of mutation steps from the human set of 9-mers, increases. The binding probability is particularly high when considering viral 9-mers that are far from all human 9-mers more than three mutation steps. A further evidence, providing significance to those special viral peptides and suggesting a potential role they can play, comes from the analysis of their distribution along viral genomes, as it revealed they are not randomly located, but they preferentially occur in specific genes. Copyright © 2018 Elsevier B.V. All rights reserved.

Functional selection of a type IV pili-binding peptide that specifically inhibits Salmonella Typhi adhesion to/invasion of human monocytic cells.

PubMed

Wu, Hong-Yan; Zhang, Xiao-Lian; Pan, Qin; Wu, Jianguo

2005-11-01

Salmonella enterica serovar Typhi (S. Typhi) is an important pathogen which infects humans exclusively and causes typhoid or enteric fever. Recently it has been discovered that type IVB pili, encoded by the S. Typhi pil operon located in the major pathogenicity island, may be important in the pathogenesis of epidemic enteric fever. To further investigate the roles of type IVB pili of S. Typhi, a 12-mer peptide (RQERSSLSKPVV), binding to the structural protein PilS of the type IVB pili of S. Typhi, was isolated with a ribosome display system. This peptide was designated as peptide R. We found that peptide R inhibited adhesion to/invasion of human monocytic THP-1 cells by piliated S. Typhi bacteria, but had no effects on nonpiliated S. Typhi bacteria. A random 12-mer peptide, of size and solubility equal to peptide R, served as a control on the specificity of peptide R. The specific interaction and binding equilibrium between the 12-mer peptide R and PilS protein was determined by isothermal titration calorimetry (ITC) and a binding constant Ka determined to be between 0.4 x 10(5) and 2.2 x 10(5)L mol(-1). Our findings suggest that the type IV pili-binding peptide R holds potential as an antibacterial peptide effective against S. Typhi infections, both in terms of prevention and therapeutic treatment. The data further provide insights into the understanding of the pathogenic roles of the type IVB pili of S. Typhi.

HLA mismatches and hematopoietic cell transplantation: structural simulations assess the impact of changes in peptide binding specificity on transplant outcome

PubMed Central

Yanover, Chen; Petersdorf, Effie W.; Malkki, Mari; Gooley, Ted; Spellman, Stephen; Velardi, Andrea; Bardy, Peter; Madrigal, Alejandro; Bignon, Jean-Denis; Bradley, Philip

2013-01-01

The success of hematopoietic cell transplantation from an unrelated donor depends in part on the degree of Human Histocompatibility Leukocyte Antigen (HLA) matching between donor and patient. We present a structure-based analysis of HLA mismatching, focusing on individual amino acid mismatches and their effect on peptide binding specificity. Using molecular modeling simulations of HLA-peptide interactions, we find evidence that amino acid mismatches predicted to perturb peptide binding specificity are associated with higher risk of mortality in a large and diverse dataset of patient-donor pairs assembled by the International Histocompatibility Working Group in Hematopoietic Cell Transplantation consortium. This analysis may represent a first step toward sequence-based prediction of relative risk for HLA allele mismatches. PMID:24482668

Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

PubMed

Sahin, Deniz; Taflan, Sevket Onur; Yartas, Gizem; Ashktorab, Hassan; Smoot, Duane T

2018-04-25

Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency of traditional techniques both in diagnosis and therapy of the disease makes the development of alternative and novel techniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be used to increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of this study is screening and identification of individual peptides specifically targeted to human gastric cancer cells using a phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptide sequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cells were used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide library were applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were established by enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clones after five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequencies of each amino acid in best binding clones to determine positively overexpressed amino acids for designing novel peptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific binding on MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acid frequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45 cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, but more importantly, data extracted from eluted phage clones may be used to design theoretical peptides with better binding properties than even experimentally selected ones

Novel ZnO-binding peptides obtained by the screening of a phage display peptide library

NASA Astrophysics Data System (ADS)

Golec, Piotr; Karczewska-Golec, Joanna; Łoś, Marcin; Węgrzyn, Grzegorz

2012-11-01

Zinc oxide (ZnO) is a semiconductor compound with a potential for wide use in various applications, including biomaterials and biosensors, particularly as nanoparticles (the size range of ZnO nanoparticles is from 2 to 100 nm, with an average of about 35 nm). Here, we report isolation of novel ZnO-binding peptides, by screening of a phage display library. Interestingly, amino acid sequences of the ZnO-binding peptides reported in this paper and those described previously are significantly different. This suggests that there is a high variability in sequences of peptides which can bind particular inorganic molecules, indicating that different approaches may lead to discovery of different peptides of generally the same activity (e.g., binding of ZnO) but having various detailed properties, perhaps crucial under specific conditions of different applications.

Semi-empirical quantum evaluation of peptide - MHC class II binding

NASA Astrophysics Data System (ADS)

González, Ronald; Suárez, Carlos F.; Bohórquez, Hugo J.; Patarroyo, Manuel A.; Patarroyo, Manuel E.

2017-01-01

Peptide presentation by the major histocompatibility complex (MHC) is a key process for triggering a specific immune response. Studying peptide-MHC (pMHC) binding from a structural-based approach has potential for reducing the costs of investigation into vaccine development. This study involved using two semi-empirical quantum chemistry methods (PM7 and FMO-DFTB) for computing the binding energies of peptides bonded to HLA-DR1 and HLA-DR2. We found that key stabilising water molecules involved in the peptide binding mechanism were required for finding high correlation with IC50 experimental values. Our proposal is computationally non-intensive, and is a reliable alternative for studying pMHC binding interactions.

Sequence-specific, nanomolar peptide binding via cucurbit[8]uril-induced folding and inclusion of neighboring side chains.

PubMed

Smith, Lauren C; Leach, David G; Blaylock, Brittney E; Ali, Omar A; Urbach, Adam R

2015-03-18

This paper describes the molecular recognition of the tripeptide Tyr-Leu-Ala by the synthetic receptor cucurbit[8]uril (Q8) in aqueous buffer with nanomolar affinity and exceptional specificity. This combination of characteristics, which also applies to antibodies, is desirable for applications in biochemistry and biotechnology but has eluded supramolecular chemists for decades. Building on prior knowledge that Q8 binds to peptides with N-terminal aromatic residues, a library screen of 105 peptides was designed to test the effects of residues adjacent to N-terminal Trp, Phe, or Tyr. The screen used tetramethylbenzobis(imidazolium) (MBBI) as a fluorescent indicator and resulted in the unexpected discovery that MBBI can serve not only as a turn-off sensor via the simultaneous inclusion of a Trp residue but also as a turn-on sensor via the competitive displacement of MBBI upon binding of Phe- or Tyr-terminated peptides. The unusual fluorescence response of the Tyr series prompted further investigation by (1)H NMR spectroscopy, electrospray ionization mass spectrometry, and isothermal titration calorimetry. From these studies, a novel binding motif was discovered in which only 1 equiv of peptide binds to Q8, and the side chains of both the N-terminal Tyr residue and its immediate neighbor bind within the Q8 cavity. For the peptide Tyr-Leu-Ala, the equilibrium dissociation constant value is 7.2 nM, whereas that of its sequence isomer Tyr-Ala-Leu is 34 μM. The high stability, recyclability, and low cost of Q8 combined with the straightforward incorporation of Tyr-Leu-Ala into recombinant proteins should make this system attractive for the development of biological applications.

Design and structure of stapled peptides binding to estrogen receptors.

PubMed

Phillips, Chris; Roberts, Lee R; Schade, Markus; Bazin, Richard; Bent, Andrew; Davies, Nichola L; Moore, Rob; Pannifer, Andrew D; Pickford, Andrew R; Prior, Stephen H; Read, Christopher M; Scott, Andrew; Brown, David G; Xu, Bin; Irving, Stephen L

2011-06-29

Synthetic peptides that specifically bind nuclear hormone receptors offer an alternative approach to small molecules for the modulation of receptor signaling and subsequent gene expression. Here we describe the design of a series of novel stapled peptides that bind the coactivator peptide site of estrogen receptors. Using a number of biophysical techniques, including crystal structure analysis of receptor-stapled peptide complexes, we describe in detail the molecular interactions and demonstrate that all-hydrocarbon staples modulate molecular recognition events. The findings have implications for the design of stapled peptides in general.

Efficient one-cycle affinity selection of binding proteins or peptides specific for a small-molecule using a T7 phage display pool.

PubMed

Takakusagi, Yoichi; Kuramochi, Kouji; Takagi, Manami; Kusayanagi, Tomoe; Manita, Daisuke; Ozawa, Hiroko; Iwakiri, Kanako; Takakusagi, Kaori; Miyano, Yuka; Nakazaki, Atsuo; Kobayashi, Susumu; Sugawara, Fumio; Sakaguchi, Kengo

2008-11-15

Here, we report an efficient one-cycle affinity selection using a natural-protein or random-peptide T7 phage pool for identification of binding proteins or peptides specific for small-molecules. The screening procedure involved a cuvette type 27-MHz quartz-crystal microbalance (QCM) apparatus with introduction of self-assembled monolayer (SAM) for a specific small-molecule immobilization on the gold electrode surface of a sensor chip. Using this apparatus, we attempted an affinity selection of proteins or peptides against synthetic ligand for FK506-binding protein (SLF) or irinotecan (Iri, CPT-11). An affinity selection using SLF-SAM and a natural-protein T7 phage pool successfully detected FK506-binding protein 12 (FKBP12)-displaying T7 phage after an interaction time of only 10 min. Extensive exploration of time-consuming wash and/or elution conditions together with several rounds of selection was not required. Furthermore, in the selection using a 15-mer random-peptide T7 phage pool and subsequent analysis utilizing receptor ligand contact (RELIC) software, a subset of SLF-selected peptides clearly pinpointed several amino-acid residues within the binding site of FKBP12. Likewise, a subset of Iri-selected peptides pinpointed part of the positive amino-acid region of residues from the Iri-binding site of the well-known direct targets, acetylcholinesterase (AChE) and carboxylesterase (CE). Our findings demonstrate the effectiveness of this method and general applicability for a wide range of small-molecules.

Biodiscovery of aluminum binding peptides

NASA Astrophysics Data System (ADS)

Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Hurley, Margaret M.; Stratis-Cullum, Dimitra

2013-05-01

Cell surface peptide display systems are large and diverse libraries of peptides (7-15 amino acids) which are presented by a display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high affinity binders to a given target of interest, and those binders quickly identified. Peptide display systems have traditionally been utilized in conjunction with organic-based targets, such as protein toxins or carbon nanotubes. However, this technology has been expanded for use with inorganic targets, such as metals, for biofabrication, hybrid material assembly and corrosion prevention. While most current peptide display systems employ viruses to host the display scaffold, we have recently shown that a bacterial host, Escherichia coli, displaying peptides in the ubiquitous, membrane protein scaffold eCPX can also provide specific peptide binders to an organic target. We have, for the first time, extended the use of this bacterial peptide display system for the biodiscovery of aluminum binding 15mer peptides. We will present the process of biopanning with macroscopic inorganic targets, binder enrichment, and binder isolation and discovery.

Increasing the affinity of selective bZIP-binding peptides through surface residue redesign.

PubMed

Kaplan, Jenifer B; Reinke, Aaron W; Keating, Amy E

2014-07-01

The coiled-coil dimer is a prevalent protein interaction motif that is important for many cellular processes. The basic leucine-zipper (bZIP) transcription factors are one family of proteins for which coiled-coil mediated dimerization is essential for function, and misregulation of bZIPs can lead to disease states including cancer. This makes coiled coils attractive protein-protein interaction targets to disrupt using engineered molecules. Previous work designing peptides to compete with native coiled-coil interactions focused primarily on designing the core residues of the interface to achieve affinity and specificity. However, folding studies on the model bZIP GCN4 show that coiled-coil surface residues also contribute to binding affinity. Here we extend a prior study in which peptides were designed to bind tightly and specifically to representative members of each of 20 human bZIP families. These "anti-bZIP" peptides were designed with an emphasis on target-binding specificity, with contributions to design-target specificity and affinity engineered considering only the coiled-coil core residues. High-throughput testing using peptide arrays indicated many successes. We have now measured the binding affinities and specificities of anti-bZIPs that bind to FOS, XBP1, ATF6, and CREBZF in solution and tested whether redesigning the surface residues can increase design-target affinity. Incorporating residues that favor helix formation into the designs increased binding affinities in all cases, providing low-nanomolar binders of each target. However, changes in surface electrostatic interactions sometimes changed the binding specificity of the designed peptides. © 2014 The Protein Society.

A specific RAGE-binding peptide biopanning from phage display random peptide library that ameliorates symptoms in amyloid β peptide-mediated neuronal disorder.

PubMed

Cai, Cuizan; Dai, Xiaoyong; Zhu, Yujie; Lian, Mengyang; Xiao, Fei; Dong, Fangyuan; Zhang, Qihao; Huang, Yadong; Zheng, Qing

2016-01-01

Alzheimer's disease (AD) is an age-related neurodegenerative disorder in which amyloid β (Aβ) peptide accumulates in the brain. The receptor for advanced glycation end product (RAGE) is a cellular binding site for Aβ peptide and mediates amyloid β-induced perturbations in cerebral vessels, neurons, and microglia in AD. Here, we identified a specific high-affinity RAGE inhibitor (APDTKTQ named RP-1) from a phage display library. RP-1 bound to RAGE and inhibited Aβ peptide-induced cellular stress in human neuroblastoma SH-SYSY cells in vitro. Three amino acids in RP-1 are identical to those in the Aβ peptide. RP-1 shows high homology to the 16-23 (KLVFFAED) regions in Aβ peptide and high-affinity RAGE. Functional analyses indicated that RP-1 significantly reduced the level of reactive oxygen species (ROS) and ROS products and that it enhanced catalase and glutathione peroxidase (GPx) activity. Furthermore, it inactivated caspase3 and caspase9 and inhibited the upregulation of RAGE, nuclear factor-κB (NF-κB), and beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) protein expression. In addition, RP-1 activated the PI3K/AKT signaling pathway, inhibiting the interaction between Bax and Bcl-2. Our data suggest that RP-1 is a potent RAGE blocker that effectively controls the progression of Aβ peptide-mediated brain disorders and that it may have potential as a disease-modifying agent for AD.

A peptide affinity column for the identification of integrin alpha IIb-binding proteins.

PubMed

Daxecker, Heide; Raab, Markus; Bernard, Elise; Devocelle, Marc; Treumann, Achim; Moran, Niamh

2008-03-01

To understand the regulation of integrin alpha(IIb)beta(3), a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and alpha-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and beta3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR.

Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor.

PubMed

Neumann, Frank; Sturm, Christine; Hülsmeyer, Martin; Dauth, Nina; Guillaume, Philippe; Luescher, Immanuel F; Pfreundschuh, Michael; Held, Gerhard

2009-08-15

In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 (103-111)/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 (103-111) peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.

Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

PubMed

Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

2017-02-01

To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

T-Epitope Designer: A HLA-peptide binding prediction server.

PubMed

Kangueane, Pandjassarame; Sakharkar, Meena Kishore

2005-05-15

The current challenge in synthetic vaccine design is the development of a methodology to identify and test short antigen peptides as potential T-cell epitopes. Recently, we described a HLA-peptide binding model (using structural properties) capable of predicting peptides binding to any HLA allele. Consequently, we have developed a web server named T-EPITOPE DESIGNER to facilitate HLA-peptide binding prediction. The prediction server is based on a model that defines peptide binding pockets using information gleaned from X-ray crystal structures of HLA-peptide complexes, followed by the estimation of peptide binding to binding pockets. Thus, the prediction server enables the calculation of peptide binding to HLA alleles. This model is superior to many existing methods because of its potential application to any given HLA allele whose sequence is clearly defined. The web server finds potential application in T cell epitope vaccine design. http://www.bioinformation.net/ted/

Specific Amyloid Binding of Polybasic Peptides In Vivo Is Retained by β-Sheet Conformers but Lost in the Disrupted Coil and All D-Amino Acid Variants.

PubMed

Wall, Jonathan S; Williams, Angela; Richey, Tina; Stuckey, Alan; Wooliver, Craig; Christopher Scott, J; Donnell, Robert; Martin, Emily B; Kennel, Stephen J

2017-10-01

The heparin-reactive, helical peptide p5 is an effective amyloid imaging agent in mice with systemic amyloidosis. Analogs of p5 with modified secondary structure characteristics exhibited altered binding to heparin, synthetic amyloid fibrils, and amyloid extracts in vitro. Herein, we further study the effects of peptide helicity and chirality on specific amyloid binding using a mouse model of systemic inflammation-associated (AA) amyloidosis. Peptides with disrupted helical structure [p5 (coil) and p5 (Pro3) ], with an extended sheet conformation [p5 (sheet) ] or an all-D enantiomer [p5 (D) ], were chemically synthesized, radioiodinated, and their biodistribution studied in WT mice as well as transgenic animals with severe systemic AA amyloidosis. Peptide binding was assessed qualitatively by using small animal single-photon emission computed tomography/x-ray computed tomography imaging and microautoradiography and quantitatively using tissue counting. Peptides with reduced helical propensity, p5 (coil) and p5 (Pro3) , exhibited significantly reduced binding to AA amyloid-laden organs. In contrast, peptide p5 (D) was retained by non-amyloid-related ligands in the liver and kidneys of both WT and AA mice, but it also bound AA amyloid in the spleen. The p5 (sheet) peptide specifically bound AA amyloid in vivo and was not retained by healthy tissues in WT animals. Modification of amyloid-targeting peptides using D-amino acids should be performed cautiously due to the introduction of unexpected secondary pharmacologic effects. Peptides that adopt a helical structure, to align charged amino acid side chains along one face, exhibit specific reactivity with amyloid; however, polybasic peptides with a propensity for β-sheet conformation are also amyloid-reactive and may yield a novel class of amyloid-targeting agents for imaging and therapy.

A Simple PB/LIE Free Energy Function Accurately Predicts the Peptide Binding Specificity of the Tiam1 PDZ Domain.

PubMed

Panel, Nicolas; Sun, Young Joo; Fuentes, Ernesto J; Simonson, Thomas

2017-01-01

PDZ domains generally bind short amino acid sequences at the C-terminus of target proteins, and short peptides can be used as inhibitors or model ligands. Here, we used experimental binding assays and molecular dynamics simulations to characterize 51 complexes involving the Tiam1 PDZ domain and to test the performance of a semi-empirical free energy function. The free energy function combined a Poisson-Boltzmann (PB) continuum electrostatic term, a van der Waals interaction energy, and a surface area term. Each term was empirically weighted, giving a Linear Interaction Energy or "PB/LIE" free energy. The model yielded a mean unsigned deviation of 0.43 kcal/mol and a Pearson correlation of 0.64 between experimental and computed free energies, which was superior to a Null model that assumes all complexes have the same affinity. Analyses of the models support several experimental observations that indicate the orientation of the α 2 helix is a critical determinant for peptide specificity. The models were also used to predict binding free energies for nine new variants, corresponding to point mutants of the Syndecan1 and Caspr4 peptides. The predictions did not reveal improved binding; however, they suggest that an unnatural amino acid could be used to increase protease resistance and peptide lifetimes in vivo . The overall performance of the model should allow its use in the design of new PDZ ligands in the future.

A Simple PB/LIE Free Energy Function Accurately Predicts the Peptide Binding Specificity of the Tiam1 PDZ Domain

PubMed Central

Panel, Nicolas; Sun, Young Joo; Fuentes, Ernesto J.; Simonson, Thomas

2017-01-01

PDZ domains generally bind short amino acid sequences at the C-terminus of target proteins, and short peptides can be used as inhibitors or model ligands. Here, we used experimental binding assays and molecular dynamics simulations to characterize 51 complexes involving the Tiam1 PDZ domain and to test the performance of a semi-empirical free energy function. The free energy function combined a Poisson-Boltzmann (PB) continuum electrostatic term, a van der Waals interaction energy, and a surface area term. Each term was empirically weighted, giving a Linear Interaction Energy or “PB/LIE” free energy. The model yielded a mean unsigned deviation of 0.43 kcal/mol and a Pearson correlation of 0.64 between experimental and computed free energies, which was superior to a Null model that assumes all complexes have the same affinity. Analyses of the models support several experimental observations that indicate the orientation of the α2 helix is a critical determinant for peptide specificity. The models were also used to predict binding free energies for nine new variants, corresponding to point mutants of the Syndecan1 and Caspr4 peptides. The predictions did not reveal improved binding; however, they suggest that an unnatural amino acid could be used to increase protease resistance and peptide lifetimes in vivo. The overall performance of the model should allow its use in the design of new PDZ ligands in the future. PMID:29018806

PepComposer: computational design of peptides binding to a given protein surface

PubMed Central

Obarska-Kosinska, Agnieszka; Iacoangeli, Alfredo; Lepore, Rosalba; Tramontano, Anna

2016-01-01

There is a wide interest in designing peptides able to bind to a specific region of a protein with the aim of interfering with a known interaction or as starting point for the design of inhibitors. Here we describe PepComposer, a new pipeline for the computational design of peptides binding to a given protein surface. PepComposer only requires the target protein structure and an approximate definition of the binding site as input. We first retrieve a set of peptide backbone scaffolds from monomeric proteins that harbor the same backbone arrangement as the binding site of the protein of interest. Next, we design optimal sequences for the identified peptide scaffolds. The method is fully automatic and available as a web server at http://biocomputing.it/pepcomposer/webserver. PMID:27131789

The Length Distribution of Class I-Restricted T Cell Epitopes Is Determined by Both Peptide Supply and MHC Allele-Specific Binding Preference.

PubMed

Trolle, Thomas; McMurtrey, Curtis P; Sidney, John; Bardet, Wilfried; Osborn, Sean C; Kaever, Thomas; Sette, Alessandro; Hildebrand, William H; Nielsen, Morten; Peters, Bjoern

2016-02-15

HLA class I-binding predictions are widely used to identify candidate peptide targets of human CD8(+) T cell responses. Many such approaches focus exclusively on a limited range of peptide lengths, typically 9 aa and sometimes 9-10 aa, despite multiple examples of dominant epitopes of other lengths. In this study, we examined whether epitope predictions can be improved by incorporating the natural length distribution of HLA class I ligands. We found that, although different HLA alleles have diverse length-binding preferences, the length profiles of ligands that are naturally presented by these alleles are much more homogeneous. We hypothesized that this is due to a defined length profile of peptides available for HLA binding in the endoplasmic reticulum. Based on this, we created a model of HLA allele-specific ligand length profiles and demonstrate how this model, in combination with HLA-binding predictions, greatly improves comprehensive identification of CD8(+) T cell epitopes. Copyright © 2016 by The American Association of Immunologists, Inc.

Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides.

PubMed

Kessler, Jan H; Mommaas, Bregje; Mutis, Tuna; Huijbers, Ivo; Vissers, Debby; Benckhuijsen, Willemien E; Schreuder, Geziena M Th; Offringa, Rienk; Goulmy, Els; Melief, Cornelis J M; van der Burg, Sjoerd H; Drijfhout, Jan W

2003-02-01

We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membrane-bound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian population. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptide-binding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules.

Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

DOE PAGES

Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; ...

2014-11-14

An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth ofmore » IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.« less

Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf

An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth ofmore » IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.« less

Binding and orientation of fibronectin on polystyrene surfaces using immobilized bacterial adhesin-related peptides.

PubMed

Klueh, U; Bryers, J D; Kreutzer, D L

2003-10-01

Fibronectin (FN) is known to bind to bacteria via high affinity receptors on bacterial surfaces known as adhesins. The binding of bacteria to FN is thought to have a key role in foreign device associated infections. For example, previous studies have indicated that Staphylococcus aureus adhesins bind to the 29 kDa NH(3) terminus end of FN, and thereby promote bacteria adherence to surfaces. Recently, the peptide sequences within the S. aureus adhesin molecule that are responsible for FN binding have been identified. Based on these observations, we hypothesize that functional FN can be bound and specifically oriented on polystyrene surfaces using bacterial adhesin-related (BRP-A) peptide. We further hypothesize that monoclonal antibodies that react with specific epitopes on the FN can be used to quantify both FN binding and orientation on these surfaces. Based on this hypothesis, we initiated a systematic investigation of the binding and orientation of FN on polystyrene surfaces using BRP-A peptide. To test this hypothesis, the binding and orientation of the FN to immobilized BRP-A was quantified using (125)I-FN, and monoclonal antibodies. (125)I-FN was used to quantitate FN binding to peptide-coated polystyrene surfaces. The orientation of bound FN was demonstrated by the use of monoclonal antibodies, which are reactive with the amine (N) or carboxyl (C) termini of the FN. The results of our studies demonstrated that when the BRP-A peptide was used to bind FN to surfaces that: 1. functional FN was bound to the peptide; 2. anti-C terminus antibodies bound to the peptide FN; and 3. only limited binding of anti-N terminus antibodies to peptide-bound FN occurred. We believe that the data that indicate an enhanced binding of anti-C antibodies reactive to anti-N antibodies are a result of the FN binding in an oriented manner with the N termini of FN bound tightly to the BRP-A on the polystyrene surface. Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 36

Engineering of the function of diamond-like carbon binding peptides through structural design.

PubMed

Gabryelczyk, Bartosz; Szilvay, Géza R; Singh, Vivek K; Mikkilä, Joona; Kostiainen, Mauri A; Koskinen, Jari; Linder, Markus B

2015-02-09

The use of phage display to select material-specific peptides provides a general route towards modification and functionalization of surfaces and interfaces. However, a rational structural engineering of the peptides for optimal affinity is typically not feasible because of insufficient structure-function understanding. Here, we investigate the influence of multivalency of diamond-like carbon (DLC) binding peptides on binding characteristics. We show that facile linking of peptides together using different lengths of spacers and multivalency leads to a tuning of affinity and kinetics. Notably, increased length of spacers in divalent systems led to significantly increased affinities. Making multimers influenced also kinetic aspects of surface competition. Additionally, the multivalent peptides were applied as surface functionalization components for a colloidal form of DLC. The work suggests the use of a set of linking systems to screen parameters for functional optimization of selected material-specific peptides.

Selection of peptides binding to metallic borides by screening M13 phage display libraries.

PubMed

Ploss, Martin; Facey, Sandra J; Bruhn, Carina; Zemel, Limor; Hofmann, Kathrin; Stark, Robert W; Albert, Barbara; Hauer, Bernhard

2014-02-10

Metal borides are a class of inorganic solids that is much less known and investigated than for example metal oxides or intermetallics. At the same time it is a highly versatile and interesting class of compounds in terms of physical and chemical properties, like semiconductivity, ferromagnetism, or catalytic activity. This makes these substances attractive for the generation of new materials. Very little is known about the interaction between organic materials and borides. To generate nanostructured and composite materials which consist of metal borides and organic modifiers it is necessary to develop new synthetic strategies. Phage peptide display libraries are commonly used to select peptides that bind specifically to metals, metal oxides, and semiconductors. Further, these binding peptides can serve as templates to control the nucleation and growth of inorganic nanoparticles. Additionally, the combination of two different binding motifs into a single bifunctional phage could be useful for the generation of new composite materials. In this study, we have identified a unique set of sequences that bind to amorphous and crystalline nickel boride (Ni3B) nanoparticles, from a random peptide library using the phage display technique. Using this technique, strong binders were identified that are selective for nickel boride. Sequence analysis of the peptides revealed that the sequences exhibit similar, yet subtle different patterns of amino acid usage. Although a predominant binding motif was not observed, certain charged amino acids emerged as essential in specific binding to both substrates. The 7-mer peptide sequence LGFREKE, isolated on amorphous Ni3B emerged as the best binder for both substrates. Fluorescence microscopy and atomic force microscopy confirmed the specific binding affinity of LGFREKE expressing phage to amorphous and crystalline Ni3B nanoparticles. This study is, to our knowledge, the first to identify peptides that bind specifically to amorphous and

Effect of solid surface charge on the binding behaviour of a metal-binding peptide

PubMed Central

Donatan, Senem; Sarikaya, Mehmet; Tamerler, Candan; Urgen, Mustafa

2012-01-01

Over the last decade, solid-binding peptides have been increasingly used as molecular building blocks coupling bio- and nanotechnology. Despite considerable research being invested in this field, the effects of many surface-related parameters that define the binding of peptide to solids are still unknown. In the quest to control biological molecules at solid interfaces and, thereby, tailoring the binding characteristics of the peptides, the use of surface charge of the solid surface may probably play an important role, which then can be used as a potential tuning parameter of peptide adsorption. Here, we report quantitative investigation on the viscoelastic properties and binding kinetics of an engineered gold-binding peptide, 3RGBP1, adsorbed onto the gold surface at different surface charge densities. The experiments were performed in aqueous solutions using an electrochemical dissipative quartz crystal microbalance system. Hydrodynamic mass, hydration state and surface coverage of the adsorbed peptide films were determined as a function of surface charge density of the gold metal substrate. Under each charged condition, binding of 3rGBP1 displayed quantitative differences in terms of adsorbed peptide amount, surface coverage ratio and hydration state. Based on the intrinsically disordered structure of the peptide, we propose a possible mechanism for binding of the peptide that can be used for tuning surface adsorption in further studies. Controlled alteration of peptide binding on solid surfaces, as shown here, may provide novel methods for surface functionalization used for bioenabled processing and fabrication of future micro- and nanodevices. PMID:22491974

Detergent enhances binding of a secreted HLA-A2 molecule to solid phase peptides.

PubMed

Tussey, L G; Frelinger, J A

1991-11-01

We have constructed a secreted analogue (sA2) of the human class I molecule HLA-A2. sA2 was affinity purified both in the presence and absence of detergent and the effects of detergent on the magnitude and specificity of A2 binding to solid phase peptides tested. sA2 purified in the presence of detergent and detergent-solubilized A2 are shown to function comparably in the binding of the synthetic peptide M.Y + 57-68, a known T-cell epitope derived from the influenza A matrix protein. The molecules binding to M.Y + 57-68 typically represent 8% to 10% of the added protein. In contrast, less than 1% of sA2 protein purified in the absence of detergent binds M.Y + 57-68. This reduced binding is not due to a change in the affinity of sA2 for M.Y + 57-68. Addition of detergent at various stages of the purification and iodination procedures indicates that the longer the sA2 molecules are exposed to detergent the better they bind. However, the concentration of detergent during the actual binding assay does not appear to be critical. We also find that while the sA2-detergent and the sA2-no detergent molecules differ in the extent to which they bind various peptides, they do not differ in their patterns of binding. We conclude that detergent probably does not influence the specificity of class I/peptide binding but does increase the number of sA2 molecules that can participate in the binding of peptide either by generating and stabilizing "empty" sA2 molecules or by stabilizing a structure that is more amenable to binding peptide.

High-throughput analysis of peptide binding modules

PubMed Central

Liu, Bernard A.; Engelmann, Brett; Nash, Piers D.

2014-01-01

Modular protein interaction domains that recognize linear peptide motifs are found in hundreds of proteins within the human genome. Some protein interaction domains such as SH2, 14-3-3, Chromo and Bromo domains serve to recognize post-translational modification of amino acids (such as phosphorylation, acetylation, methylation etc.) and translate these into discrete cellular responses. Other modules such as SH3 and PDZ domains recognize linear peptide epitopes and serve to organize protein complexes based on localization and regions of elevated concentration. In both cases, the ability to nucleate specific signaling complexes is in large part dependent on the selectivity of a given protein module for its cognate peptide ligand. High throughput analysis of peptide-binding domains by peptide or protein arrays, phage display, mass spectrometry or other HTP techniques provides new insight into the potential protein-protein interactions prescribed by individual or even whole families of modules. Systems level analyses have also promoted a deeper understanding of the underlying principles that govern selective protein-protein interactions and how selectivity evolves. Lastly, there is a growing appreciation for the limitations and potential pitfalls of high-throughput analysis of protein-peptide interactomes. This review will examine some of the common approaches utilized for large-scale studies of protein interaction domains and suggest a set of standards for the analysis and validation of datasets from large-scale studies of peptide-binding modules. We will also highlight how data from large-scale studies of modular interaction domain families can provide insight into systems level properties such as the linguistics of selective interactions. PMID:22610655

Identification and Characterization of Strychnine-Binding Peptides Using Phage-Display Screening.

PubMed

Zhang, Fang; Wang, Min; Qiu, Zheng; Wang, Xiao-Meng; Xu, Chun-Lei; Zhang, Xia

2017-01-01

In drug development, phage display is a high-throughput method for identifying the specific cellular targets of drugs. However, insoluble small chemicals remain intractable to this technique because of the difficulty of presenting molecules to phages without occupying or destroying the limited functional groups. In the present study, we selected Strychnine (Stry) as a model compounda and sought to develope an alternative in vitro biopanning strategy against insoluble suspension. A phage library displaying random sequences of fifteen peptides was employed to screen for interactions between Stry and its cellular selective binding peptides, which are of great value to have a complete understanding of the mechanism of Stry for its antitumor activity. After four rounds of biopanning, a selection of 100 binding clones was randomly picked and subjected to modified proliferation and diffusion assays to evaluate the binding affinity of the clones. Finally, eleven clones were identified as positive binders. The corresponding peptides were synthesized and detected for their binding activities using surface plasmon resonance imaging (SPRi). Our study provides a feasible scheme for confirming the interaction of chemical compounds and cellular binding peptides. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Selective Photoaffinity Labeling Identifies the Signal Peptide Binding Domain on SecA

PubMed Central

Musial-Siwek, Monika; Rusch, Sharyn L.; Kendall, Debra A.

2007-01-01

SecA, an ATPase crucial to the Sec-dependent translocation machinery in Escherichia coli, recognizes and directly binds the N-terminal signal peptide of an exported preprotein. This interaction plays a central role in the targeting and transport of preproteins via the SecYEG channel. Here we identify the Signal Peptide Binding Groove (SPBG) on SecA addressing a key issue regarding the SecA-preprotein interaction. We employ a synthetic signal peptide containing the photoreactive benzoylphenylalanine to efficiently and specifically label SecA containing a unique Factor Xa site. Comparison of the photolabeled fragment from the subsequent proteolysis of several SecAs, which vary only in the location of the Factor Xa site, reveals one 53-residue segment in common with the entire series. The covalently modified SecA segment produced is the same in aqueous solution and in lipid vesicles. This spans amino acids 269 to 322 of the E. coli protein, which is distinct from a previously proposed signal peptide binding site, and contributes to a hydrophobic peptide binding groove evident in molecular models of SecA. PMID:17084862

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soykut, Esra Acar; Dudak, Fahriye Ceyda; Boyaci, Ismail Hakki

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinitymore » to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 {+-} 0.7 x 10{sup 5} M{sup -1} which indicates a strong binding close to that of antibody.« less

Reversibly Switchable, pH-Dependent Peptide Ligand Binding via 3,5-Diiodotyrosine Substitutions.

PubMed

Ngambenjawong, Chayanon; Sylvestre, Meilyn; Gustafson, Heather H; Pineda, Julio Marco B; Pun, Suzie H

2018-04-20

Cell type-specific targeting ligands utilized in drug delivery applications typically recognize receptors that are overexpressed on the cells of interest. Nonetheless, these receptors may also be expressed, to varying extents, on off-target cells, contributing to unintended side effects. For the selectivity profile of targeting ligands in cancer therapy to be improved, stimuli-responsive masking of these ligands with acid-, redox-, or enzyme-cleavable molecules has been reported, whereby the targeting ligands are exposed in specific environments, e.g., acidic tumor hypoxia. One possible drawback of these systems lies in their one-time, permanent trigger, which enables the "demasked" ligands to bind off-target cells if released back into the systemic circulation. A promising strategy to address the aforementioned problem is to design ligands that show selective binding based on ionization state, which may be microenvironment-dependent. In this study, we report a systematic strategy to engineer low pH-selective targeting peptides using an M2 macrophage-targeting peptide (M2pep) as an example. 3,5-Diiodotyrosine mutagenesis into native tyrosine residues of M2pep confers pH-dependent binding behavior specific to acidic environment (pH 6) when the amino acid is protonated into the native tyrosine-like state. At physiological pH of 7.4, the hydroxyl group of 3,5-diiodotyrosine on the peptide is deprotonated leading to interruption of the peptide native binding property. Our engineered pH-responsive M2pep (Ac-Y-Î-Î) binds target M2 macrophages more selectively at pH 6 than at pH 7.4. In addition, 3,5-diiodotyrosine substitutions also improve serum stability of the peptide. Finally, we demonstrate pH-dependent reversibility in target binding via a postbinding peptide elution study. The strategy presented here should be applicable for engineering pH-dependent functionality of other targeting peptides with potential applications in physiology-dependent in vivo targeting

Understanding and predicting binding between human leukocyte antigens (HLAs) and peptides by network analysis.

PubMed

Luo, Heng; Ye, Hao; Ng, Hui; Shi, Leming; Tong, Weida; Mattes, William; Mendrick, Donna; Hong, Huixiao

2015-01-01

As the major histocompatibility complex (MHC), human leukocyte antigens (HLAs) are one of the most polymorphic genes in humans. Patients carrying certain HLA alleles may develop adverse drug reactions (ADRs) after taking specific drugs. Peptides play an important role in HLA related ADRs as they are the necessary co-binders of HLAs with drugs. Many experimental data have been generated for understanding HLA-peptide binding. However, efficiently utilizing the data for understanding and accurately predicting HLA-peptide binding is challenging. Therefore, we developed a network analysis based method to understand and predict HLA-peptide binding. Qualitative Class I HLA-peptide binding data were harvested and prepared from four major databases. An HLA-peptide binding network was constructed from this dataset and modules were identified by the fast greedy modularity optimization algorithm. To examine the significance of signals in the yielded models, the modularity was compared with the modularity values generated from 1,000 random networks. The peptides and HLAs in the modules were characterized by similarity analysis. The neighbor-edges based and unbiased leverage algorithm (Nebula) was developed for predicting HLA-peptide binding. Leave-one-out (LOO) validations and two-fold cross-validations were conducted to evaluate the performance of Nebula using the constructed HLA-peptide binding network. Nine modules were identified from analyzing the HLA-peptide binding network with a highest modularity compared to all the random networks. Peptide length and functional side chains of amino acids at certain positions of the peptides were different among the modules. HLA sequences were module dependent to some extent. Nebula archived an overall prediction accuracy of 0.816 in the LOO validations and average accuracy of 0.795 in the two-fold cross-validations and outperformed the method reported in the literature. Network analysis is a useful approach for analyzing large and

Understanding and predicting binding between human leukocyte antigens (HLAs) and peptides by network analysis

PubMed Central

2015-01-01

Background As the major histocompatibility complex (MHC), human leukocyte antigens (HLAs) are one of the most polymorphic genes in humans. Patients carrying certain HLA alleles may develop adverse drug reactions (ADRs) after taking specific drugs. Peptides play an important role in HLA related ADRs as they are the necessary co-binders of HLAs with drugs. Many experimental data have been generated for understanding HLA-peptide binding. However, efficiently utilizing the data for understanding and accurately predicting HLA-peptide binding is challenging. Therefore, we developed a network analysis based method to understand and predict HLA-peptide binding. Methods Qualitative Class I HLA-peptide binding data were harvested and prepared from four major databases. An HLA-peptide binding network was constructed from this dataset and modules were identified by the fast greedy modularity optimization algorithm. To examine the significance of signals in the yielded models, the modularity was compared with the modularity values generated from 1,000 random networks. The peptides and HLAs in the modules were characterized by similarity analysis. The neighbor-edges based and unbiased leverage algorithm (Nebula) was developed for predicting HLA-peptide binding. Leave-one-out (LOO) validations and two-fold cross-validations were conducted to evaluate the performance of Nebula using the constructed HLA-peptide binding network. Results Nine modules were identified from analyzing the HLA-peptide binding network with a highest modularity compared to all the random networks. Peptide length and functional side chains of amino acids at certain positions of the peptides were different among the modules. HLA sequences were module dependent to some extent. Nebula archived an overall prediction accuracy of 0.816 in the LOO validations and average accuracy of 0.795 in the two-fold cross-validations and outperformed the method reported in the literature. Conclusions Network analysis is a

Inorganic binding peptides designed by phage display techniques for biotechnology applications

NASA Astrophysics Data System (ADS)

Liao, Chih-Wei

Biomacromolecules play an important role in the control of hard tissue structure and function via specific molecular recognition interactions between proteins of the matrix and inorganic species of the biomineral phase. During the construction of the tissue, biomacromolecules are usually folded into a certain comformation, analogous to a "lock" for fitting with other proteins or smaller molecules as a "key". Currently, the rational design of molecular recognition in biomacro-molecules is still hard to accomplish because the protein conformation is too complex to precisely predict based on the existing conformational information of proteins found in biological systems. In the past two decades, the combinatorial approach (e.g. phage display techniques) has been used to select short binding peptides with molecular recognition to an inorganic target material without a prior knowledge of the amino acid sequence required for the specific binding. The technique has been referred to as "biopanning" because bacteriophages are used to "screen" for peptides that exhibit strong binding to a target material of interest. In this study, two diverse applications were chosen to demonstrate the utility of the biopanning approach. In one project, phage display techniques were used to pan for Indium Zinc Oxide (InZnO) binding peptides to serve as linkers between transducer devices and biosensing elements for demonstration of the feasibility of reversibly electro-activated biosensors. The amorphous InZnO, with its homogeneous surface, led to three consensus peptide sequences, AGFPNSTHSSNL, SHAPDSTWFALF, and TNSSSQFVVAIP. In addition, it was demonstrated that some selected phage clones of the InZnO binding peptides were able to be released from the InZnO surface after applying a voltage of 1400 mV on an electro-activated releasing device. In the second project, phage display techniques were used to select phage clones that bind specifically to francolite mineral in order to achieve

Genome-Wide Prediction and Validation of Peptides That Bind Human Prosurvival Bcl-2 Proteins

PubMed Central

DeBartolo, Joe; Taipale, Mikko; Keating, Amy E.

2014-01-01

Programmed cell death is regulated by interactions between pro-apoptotic and prosurvival members of the Bcl-2 family. Pro-apoptotic family members contain a weakly conserved BH3 motif that can adopt an alpha-helical structure and bind to a groove on prosurvival partners Bcl-xL, Bcl-w, Bcl-2, Mcl-1 and Bfl-1. Peptides corresponding to roughly 13 reported BH3 motifs have been verified to bind in this manner. Due to their short lengths and low sequence conservation, BH3 motifs are not detected using standard sequence-based bioinformatics approaches. Thus, it is possible that many additional proteins harbor BH3-like sequences that can mediate interactions with the Bcl-2 family. In this work, we used structure-based and data-based Bcl-2 interaction models to find new BH3-like peptides in the human proteome. We used peptide SPOT arrays to test candidate peptides for interaction with one or more of the prosurvival proteins Bcl-xL, Bcl-w, Bcl-2, Mcl-1 and Bfl-1. For the 36 most promising array candidates, we quantified binding to all five human receptors using direct and competition binding assays in solution. All 36 peptides showed evidence of interaction with at least one prosurvival protein, and 22 peptides bound at least one prosurvival protein with a dissociation constant between 1 and 500 nM; many peptides had specificity profiles not previously observed. We also screened the full-length parent proteins of a subset of array-tested peptides for binding to Bcl-xL and Mcl-1. Finally, we used the peptide binding data, in conjunction with previously reported interactions, to assess the affinity and specificity prediction performance of different models. PMID:24967846

Novel Peptide with Specific Calcium-Binding Capacity from Schizochytrium sp. Protein Hydrolysates and Calcium Bioavailability in Caco-2 Cells

PubMed Central

Cai, Xixi; Lin, Jiaping; Wang, Shaoyun

2016-01-01

Peptide-calcium can probably be a suitable supplement to improve calcium absorption in the human body. In this study, a specific peptide Phe-Tyr (FY) with calcium-binding capacity was purified from Schizochytrium sp. protein hydrolysates through gel filtration chromatography and reversed phase HPLC. The calcium-binding capacity of FY reached 128.77 ± 2.57 μg/mg. Results of ultraviolet spectroscopy, fluorescence spectroscopy, and infrared spectroscopy showed that carboxyl groups, amino groups, and amido groups were the major chelating sites. FY-Ca exhibited excellent thermal stability and solubility, which were beneficial to be absorbed and transported in the basic intestinal tract of the human body. Moreover, the calcium bioavailability in Caco-2 cells showed that FY-Ca could enhance calcium uptake efficiency by more than three times when compared with CaCl2, and protect calcium ions against dietary inhibitors, such as tannic acid, oxalate, phytate, and Zn2+. Our findings further the progress of algae-based peptide-calcium, suggesting that FY-Ca has the potential to be developed as functionally nutraceutical additives. PMID:28036002

Phage display selection of peptides that target calcium-binding proteins.

PubMed

Vetter, Stefan W

2013-01-01

Phage display allows to rapidly identify peptide sequences with binding affinity towards target proteins, for example, calcium-binding proteins (CBPs). Phage technology allows screening of 10(9) or more independent peptide sequences and can identify CBP binding peptides within 2 weeks. Adjusting of screening conditions allows selecting CBPs binding peptides that are either calcium-dependent or independent. Obtained peptide sequences can be used to identify CBP target proteins based on sequence homology or to quickly obtain peptide-based CBP inhibitors to modulate CBP-target interactions. The protocol described here uses a commercially available phage display library, in which random 12-mer peptides are displayed on filamentous M13 phages. The library was screened against the calcium-binding protein S100B.

A Review on Recent Patents and Applications of Inorganic Material Binding Peptides.

PubMed

Thota, Veeranjaneyulu; Perry, Carole C

2017-01-01

Although the popularity of using combinatorial display techniques for recognising unique peptides having high affinity for inorganic (nano) particles has grown rapidly, there are no systematic reviews showcasing current developments or patents on binding peptides specific to these materials. In this review, we summarize and discuss recent progress in patents on material binding peptides specifically exploring inorganic nano surfaces such as metals, metal oxides, minerals, carbonbased materials, polymer based materials, magnetic materials and semiconductors. We consider both the peptide display strategies used and the exploitation of the identified peptides in the generation of advanced nanomaterials. In order to get a clear picture on the number of patents and literature present to date relevant to inorganic material binding biomolecules and their applications, a thorough online search was conducted using national and worldwide databases. The literature search include standard bibliographic databases while patents included EPO Espacenet, WIPO patent scope, USPTO, Google patent search, Patent lens, etc. along with commercial databases such as Derwent and Patbase. Both English and American spellings were included in the searches. The initial number of patents found related to material binders were 981. After reading and excluding irrelevant patents such as organic binding peptides, works published before 2001, repeated patents, documents not in English etc., 51 highly relevant patents published from 2001 onwards were selected and analysed. These patents were further separated into six categories based on their target inorganic material and combinatorial library used. They include relevant patents on metal, metal oxide or combination binding peptides (19), magnetic and semiconductor binding peptides (8), carbon based (3), mineral (5), polymer (8) and other binders (9). Further, how these material specific binders have been used to synthesize simple to complex bio- or

Binding and thermodynamics of REV peptide-ctDNA interaction.

PubMed

Upadhyay, Santosh Kumar

2017-03-01

The thermodynamics of DNA-ligand binding is important as it provides useful information to understand the details of binding processes. HIV-1 REV response element (RRE) located in the env coding region of the viral genome is reported to be well conserved across different HIV-1 isolates. In this study, the binding characteristics of Calf thymus DNA (ctDNA) and REV peptide from HIV-1 were investigated using spectroscopic (UV-visible, fluorescence, and circular dichroism (CD)) and isothermal titration calorimetric (ITC) techniques. Thermal stability and ligand binding properties of the ctDNA revealed that native ctDNA had a T m of 75.5 °C, whereas the ctDNA-REV peptide complex exhibited an incremental shift in the T m by 8 °C, indicating thermal stability of the complex. CD data indicated increased ellipticity due to large conformational changes in ctDNA molecule upon binding with REV peptide and two binding stoichiometric modes are apparent. The ctDNA experienced condensation due to large conformational changes in the presence of REV peptide and positive B→Ψ transition was observed at higher molar charge ratios. Fluorescence studies performed at several ligand concentrations revealed a gradual decrease in the fluorescence intensity of EtBr-bound ctDNA in response to increasing ligand concentrations. The fluorescence data further confirmed two stoichiometric modes of binding for ctDNA-REV peptide complex as previously observed with CD studies. The binding enthalpies were determined using ITC in the temperature range of 293 K-308 K. The ITC binding isotherm was exothermic at all temperatures examined, with low ΔH values indicating that the ctDNA-REV peptide interaction is driven largely by entropy. The heat capacity change (ΔC p ) was insignificant, an unusual finding in the area of DNA-peptide interaction studies. The variation in the values obtained for ΔH, ΔS, and ΔG with temperature further suggests that ctDNA-REV peptide interaction is entropically

Chemokine CCR3 ligands-binding peptides derived from a random phage-epitope library.

PubMed

Houimel, Mehdi; Mazzucchelli, Luca

2013-01-01

Eosinophils are major effectors cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The human chemokine receptor C-C receptor 3 (hCCR3) provides a mechanism for the recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop peptides antagonists of hCCR3-hCCL11 (human eotaxin) interactions, a random bacteriophage hexapeptide library was used to map structural features of hCCR3 by determining the epitopes of neutralizing anti-hCCR3 mAb 7B11. This mAb t is selective for hCCR3 and exhibit potent antagonist activity in receptor binding and functional assays. After three rounds of biopanning, four mAb7B11-binding peptides were identified from a 6-mer linear peptide library. The phage bearing the peptides showed specific binding to immobilized mAb 7B11 with over 94% of phages bound being competitively inhibited by free synthetic peptides. In FACScan analysis all selected phage peptides were able to strongly inhibit the binding of mAb 7B11 to hCCR3-transfected preB-300-19 murine cells. Furthermore, synthetic peptides of the corresponding phage epitopes were effective in blocking the antibody-hCCR3 interactions and to inhibit the binding of hCCL11 to hCCR3 transfectants. Chemically synthesized peptides CKGERF, FERKGK, SSMKVK and RHVSSQ, effectively competed for (125)I-hCCL11 binding to hCCR3 with IC(50) ranging from 3.5 to 9.7μM. Calcium release and chemotaxis of hCCR3 transfectants or human eosinophils were inhibited by all peptides in a dose-dependent manner. Furthermore, they showed inhibitory effects on chemotaxis of human eosinophils induced by hCCL11, hCCL5, hCCL7, hCCL8, and hCCL24. Specificities of all selected peptides were assessed with hCXCR1, hCXCR2, hCXCR3, and hCCR5 receptors. Peptides CKGERF and FERKGK showed inhibitory effects on eosinophil chemotaxis in a murine model of mCCL11-induced

Using peptide array to identify binding motifs and interaction networks for modular domains.

PubMed

Li, Shawn S-C; Wu, Chenggang

2009-01-01

Specific protein-protein interactions underlie all essential biological processes and form the basis of cellular signal transduction. The recognition of a short, linear peptide sequence in one protein by a modular domain in another represents a common theme of macromolecular recognition in cells, and the importance of this mode of protein-protein interaction is highlighted by the large number of peptide-binding domains encoded by the human genome. This phenomenon also provides a unique opportunity to identify protein-protein binding events using peptide arrays and complementary biochemical assays. Accordingly, high-density peptide array has emerged as a useful tool by which to map domain-mediated protein-protein interaction networks at the proteome level. Using the Src-homology 2 (SH2) and 3 (SH3) domains as examples, we describe the application of oriented peptide array libraries in uncovering specific motifs recognized by an SH2 domain and the use of high-density peptide arrays in identifying interaction networks mediated by the SH3 domain. Methods reviewed here could also be applied to other modular domains, including catalytic domains, that recognize linear peptide sequences.

Binding stability of peptides on major histocompatibility complex class I proteins: role of entropy and dynamics.

PubMed

Gul, Ahmet; Erman, Burak

2018-01-16

Prediction of peptide binding on specific human leukocyte antigens (HLA) has long been studied with successful results. We herein describe the effects of entropy and dynamics by investigating the binding stabilities of 10 nanopeptides on various HLA Class I alleles using a theoretical model based on molecular dynamics simulations. The fluctuational entropies of the peptides are estimated over a temperature range of 310-460 K. The estimated entropies correlate well with experimental binding affinities of the peptides: peptides that have higher binding affinities have lower entropies compared to non-binders, which have significantly larger entropies. The computation of the entropies is based on a simple model that requires short molecular dynamics trajectories and allows for approximate but rapid determination. The paper draws attention to the long neglected dynamic aspects of peptide binding, and provides a fast computation scheme that allows for rapid scanning of large numbers of peptides on selected HLA antigens, which may be useful in defining the right peptides for personal immunotherapy.

Binding stability of peptides on major histocompatibility complex class I proteins: role of entropy and dynamics

NASA Astrophysics Data System (ADS)

Gul, Ahmet; Erman, Burak

2018-03-01

Prediction of peptide binding on specific human leukocyte antigens (HLA) has long been studied with successful results. We herein describe the effects of entropy and dynamics by investigating the binding stabilities of 10 nanopeptides on various HLA Class I alleles using a theoretical model based on molecular dynamics simulations. The fluctuational entropies of the peptides are estimated over a temperature range of 310-460 K. The estimated entropies correlate well with experimental binding affinities of the peptides: peptides that have higher binding affinities have lower entropies compared to non-binders, which have significantly larger entropies. The computation of the entropies is based on a simple model that requires short molecular dynamics trajectories and allows for approximate but rapid determination. The paper draws attention to the long neglected dynamic aspects of peptide binding, and provides a fast computation scheme that allows for rapid scanning of large numbers of peptides on selected HLA antigens, which may be useful in defining the right peptides for personal immunotherapy.

A computational method for selecting short peptide sequences for inorganic material binding.

PubMed

Nayebi, Niloofar; Cetinel, Sibel; Omar, Sara Ibrahim; Tuszynski, Jack A; Montemagno, Carlo

2017-11-01

Discovering or designing biofunctionalized materials with improved quality highly depends on the ability to manipulate and control the peptide-inorganic interaction. Various peptides can be used as assemblers, synthesizers, and linkers in the material syntheses. In another context, specific and selective material-binding peptides can be used as recognition blocks in mining applications. In this study, we propose a new in silico method to select short 4-mer peptides with high affinity and selectivity for a given target material. This method is illustrated with the calcite (104) surface as an example, which has been experimentally validated. A calcite binding peptide can play an important role in our understanding of biomineralization. A practical aspect of calcite is a need for it to be selectively depressed in mining sites. © 2017 Wiley Periodicals, Inc.

Screening and Identification of a Phage Display Derived Peptide That Specifically Binds to the CD44 Protein Region Encoded by Variable Exons.

PubMed

Zhang, Dan; Jia, Huan; Li, Weiming; Hou, Yingchun; Lu, Shaoying; He, Shuixiang

2016-01-01

CD44, especially the isoforms with variable exons (CD44v), is a promising biomarker for the detection of cancer. To develop a CD44v-specific probe, we screened a 7-mer phage peptide library against the CD44v3-v10 protein using an improved subtractive method. The consensus sequences with the highest frequency (designated CV-1) emerged after four rounds of panning. The binding affinity and specificity of the CV-1 phage and the synthesized peptide for the region of CD44 encoded by the variable exons were confirmed using enzyme-linked immunosorbent assay and competitive inhibition assays. Furthermore, the binding of the CV-1 probe to gastric cancer cells and tissues was validated using immunofluorescence and immunohistochemistry assays. CV-1 sensitively and specifically bound to CD44v on cancer cells and tissues. Thus, CV-1 has the potential to serve as a promising probe for cancer molecular imaging and target therapy. © 2015 Society for Laboratory Automation and Screening.

Biogenic and Synthetic Peptides with Oppositely Charged Amino Acids as Binding Sites for Mineralization.

PubMed

Lemloh, Marie-Louise; Altintoprak, Klara; Wege, Christina; Weiss, Ingrid M; Rothenstein, Dirk

2017-01-28

Proteins regulate diverse biological processes by the specific interaction with, e.g., nucleic acids, proteins and inorganic molecules. The generation of inorganic hybrid materials, such as shell formation in mollusks, is a protein-controlled mineralization process. Moreover, inorganic-binding peptides are attractive for the bioinspired mineralization of non-natural inorganic functional materials for technical applications. However, it is still challenging to identify mineral-binding peptide motifs from biological systems as well as for technical systems. Here, three complementary approaches were combined to analyze protein motifs consisting of alternating positively and negatively charged amino acids: (i) the screening of natural biomineralization proteins; (ii) the selection of inorganic-binding peptides derived from phage display; and (iii) the mineralization of tobacco mosaic virus (TMV)-based templates. A respective peptide motif displayed on the TMV surface had a major impact on the SiO₂ mineralization. In addition, similar motifs were found in zinc oxide- and zirconia-binding peptides indicating a general binding feature. The comparative analysis presented here raises new questions regarding whether or not there is a common design principle based on acidic and basic amino acids for peptides interacting with minerals.

Characterizing SH2 Domain Specificity and Network Interactions Using SPOT Peptide Arrays.

PubMed

Liu, Bernard A

2017-01-01

Src Homology 2 (SH2) domains are protein interaction modules that recognize and bind tyrosine phosphorylated ligands. Their ability to distinguish binding to over thousands of potential phosphotyrosine (pTyr) ligands within the cell is critical for the fidelity of receptor tyrosine kinase (RTK) signaling. Within humans there are over a hundred SH2 domains with more than several thousand potential ligands across many cell types and cell states. Therefore, defining the specificity of individual SH2 domains is critical for predicting and identifying their physiological ligands. Here, in this chapter, I describe the broad use of SPOT peptide arrays for examining SH2 domain specificity. An orientated peptide array library (OPAL) approach can uncover both favorable and non-favorable residues, thus providing an in-depth analysis to SH2 specificity. Moreover, I discuss the application of SPOT arrays for paneling SH2 ligand binding with physiological peptides.

Glucagon-like peptide-1 binding to rat skeletal muscle.

PubMed

Delgado, E; Luque, M A; Alcántara, A; Trapote, M A; Clemente, F; Galera, C; Valverde, I; Villanueva-Peñacarrillo, M L

1995-01-01

We have found [125I]glucagon-like peptide-1(7-36)-amide-specific binding activity in rat skeletal muscle plasma membranes, with an estimated M(r) of 63,000 by cross-linking and SDS-PAGE. The specific binding was time and membrane protein concentration dependent, and displaceable by unlabeled GLP-1(7-36)-amide with an ID50 of 3 x 10(-9) M of the peptide; GLP-1(1-36)-amide also competed, whereas glucagon and insulin did not. GLP-1(7-36)-amide did not modify the basal adenylate cyclase activity in skeletal muscle plasma membranes. These data, together with our previous finding of a potent glycogenic effect of GLP-1(7-36)-amide in rat soleus muscle, and also in isolated hepatocytes, which was not accompanied by a rise in the cell cyclic AMP content, lead use to believe that the insulin-like effects of this peptide on glucose metabolism in the muscle could be mediated by a type of receptor somehow different to that described for GLP-1 in pancreatic B cells, where GLP-1 action is mediated by the cyclic AMP-adenylate cyclase system.

EDB Fibronectin Specific Peptide for Prostate Cancer Targeting.

PubMed

Han, Zheng; Zhou, Zhuxian; Shi, Xiaoyue; Wang, Junpeng; Wu, Xiaohui; Sun, Da; Chen, Yinghua; Zhu, Hui; Magi-Galluzzi, Cristina; Lu, Zheng-Rong

2015-05-20

Extradomain-B fibronectin (EDB-FN), one of the oncofetal fibronectin (onfFN) isoforms, is a high-molecular-weight glycoprotein that mediates cell adhesion and migration. The expression of EDB-FN is associated with a number of cancer-related biological processes such as tumorigenesis, angiogenesis, and epithelial-to-mesenchymal transition (EMT). Here, we report the development of a small peptide specific to EDB-FN for targeting prostate cancer. A cyclic nonapeptide, CTVRTSADC (ZD2), was identified using peptide phage display. A ZD2-Cy5 conjugate was synthesized to accomplish molecular imaging of prostate cancer in vitro and in vivo. ZD2-Cy5 demonstrated effective binding to up-regulated EDB-FN secreted by TGF-β-induced PC3 cancer cells following EMT. Following intravenous injections, the targeted fluorescent probe specifically bound to and delineated PC3-GFP prostate tumors in nude mice bearing the tumor xenografts. ZD2-Cy5 also showed stronger binding to human prostate tumor specimens with a higher Gleason score (GS9) compared to those with a lower score (GS 7), with no binding in benign prostatic hyperplasia (BPH). Thus, the ZD2 peptide is a promising strategy for molecular imaging and targeted therapy of prostate cancer.

Fast and reliable prediction of domain-peptide binding affinity using coarse-grained structure models.

PubMed

Tian, Feifei; Tan, Rui; Guo, Tailin; Zhou, Peng; Yang, Li

2013-07-01

Domain-peptide recognition and interaction are fundamentally important for eukaryotic signaling and regulatory networks. It is thus essential to quantitatively infer the binding stability and specificity of such interaction based upon large-scale but low-accurate complex structure models which could be readily obtained from sophisticated molecular modeling procedure. In the present study, a new method is described for the fast and reliable prediction of domain-peptide binding affinity with coarse-grained structure models. This method is designed to tolerate strong random noises involved in domain-peptide complex structures and uses statistical modeling approach to eliminate systematic bias associated with a group of investigated samples. As a paradigm, this method was employed to model and predict the binding behavior of various peptides to four evolutionarily unrelated peptide-recognition domains (PRDs), i.e. human amph SH3, human nherf PDZ, yeast syh GYF and yeast bmh 14-3-3, and moreover, we explored the molecular mechanism and biological implication underlying the binding of cognate and noncognate peptide ligands to their domain receptors. It is expected that the newly proposed method could be further used to perform genome-wide inference of domain-peptide binding at three-dimensional structure level. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Learning a peptide-protein binding affinity predictor with kernel ridge regression

PubMed Central

2013-01-01

Background The cellular function of a vast majority of proteins is performed through physical interactions with other biomolecules, which, most of the time, are other proteins. Peptides represent templates of choice for mimicking a secondary structure in order to modulate protein-protein interaction. They are thus an interesting class of therapeutics since they also display strong activity, high selectivity, low toxicity and few drug-drug interactions. Furthermore, predicting peptides that would bind to a specific MHC alleles would be of tremendous benefit to improve vaccine based therapy and possibly generate antibodies with greater affinity. Modern computational methods have the potential to accelerate and lower the cost of drug and vaccine discovery by selecting potential compounds for testing in silico prior to biological validation. Results We propose a specialized string kernel for small bio-molecules, peptides and pseudo-sequences of binding interfaces. The kernel incorporates physico-chemical properties of amino acids and elegantly generalizes eight kernels, comprised of the Oligo, the Weighted Degree, the Blended Spectrum, and the Radial Basis Function. We provide a low complexity dynamic programming algorithm for the exact computation of the kernel and a linear time algorithm for it’s approximation. Combined with kernel ridge regression and SupCK, a novel binding pocket kernel, the proposed kernel yields biologically relevant and good prediction accuracy on the PepX database. For the first time, a machine learning predictor is capable of predicting the binding affinity of any peptide to any protein with reasonable accuracy. The method was also applied to both single-target and pan-specific Major Histocompatibility Complex class II benchmark datasets and three Quantitative Structure Affinity Model benchmark datasets. Conclusion On all benchmarks, our method significantly (p-value ≤ 0.057) outperforms the current state-of-the-art methods at predicting

Non-ionic detergents facilitate non-specific binding of M13 bacteriophage to polystyrene surfaces.

PubMed

Hakami, Abdulrahim R; Ball, Jonathan K; Tarr, Alexander W

2015-09-01

Phage-displayed random peptide libraries are widely used for identifying peptide interactions with proteins and other substrates. Selection of peptide ligands involves iterative rounds of affinity enrichment. The binding properties of the selected phage clones are routinely tested using immunoassay after propagation to high titre in a bacterial host and precipitation using polyethylene glycol (PEG) and high salt concentration. These immunoassays can suffer from low sensitivity and high background signals. Polysorbate 20 (Tween(®) 20) is a non-ionic detergent commonly used in immunoassay washing buffers to reduce non-specific binding, and is also used as a blocking reagent. We have observed that Tween 20 enhances non-specific M13 library phage binding in a peptide-independent manner. Other non-ionic detergents were also found to promote significant, dose-dependent non-specific phage binding in ELISA. This effect was not observed for assays using phage concentrated by ultracentrifugation, suggesting that interactions occur between detergents and the PEG-precipitated phage, irrespective of the displayed peptide motif. This artefact may impact on successful affinity selection of peptides from phage-display libraries. We propose alternative methods for screening phage libraries for identifying binding interactions with target ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

Direct and selective immobilization of proteins by means of an inorganic material-binding peptide: discussion on functionalization in the elongation to material-binding peptide.

PubMed

Yokoo, Nozomi; Togashi, Takanari; Umetsu, Mitsuo; Tsumoto, Kouhei; Hattori, Takamitsu; Nakanishi, Takeshi; Ohara, Satoshi; Takami, Seiichi; Naka, Takashi; Abe, Hiroya; Kumagai, Izumi; Adschiri, Tadafumi

2010-01-14

Using an artificial peptide library, we have identified a peptide with affinity for ZnO materials that could be used to selectively accumulate ZnO particles on polypropylene-gold plates. In this study, we fused recombinant green fluorescent protein (GFP) with this ZnO-binding peptide (ZnOBP) and then selectively immobilized the fused protein on ZnO particles. We determined an appropriate condition for selective immobilization of recombinant GFP, and the ZnO-binding function of ZnOBP-fused GFP was examined by elongating the ZnOBP tag from a single amino acid to the intact sequence. The fusion of ZnOBP with GFP enabled specific adsorption of GFP on ZnO substrates in an appropriate solution, and thermodynamic studies showed a predominantly enthalpy-dependent electrostatic interaction between ZnOBP and the ZnO surface. The ZnOBP's binding affinity for the ZnO surface increased first in terms of material selectivity and then in terms of high affinity as the GFP-fused peptide was elongated from a single amino acid to intact ZnOBP. We concluded that the enthalpy-dependent interaction between ZnOBP and ZnO was influenced by the presence of not only charged amino acids but also their surrounding residues in the ZnOBP sequence.

BiPPred: Combined sequence- and structure-based prediction of peptide binding to the Hsp70 chaperone BiP.

PubMed

Schneider, Markus; Rosam, Mathias; Glaser, Manuel; Patronov, Atanas; Shah, Harpreet; Back, Katrin Christiane; Daake, Marina Angelika; Buchner, Johannes; Antes, Iris

2016-10-01

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi-scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence-based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence-based prediction models were fitted using this and other peptide binding data. A structure-based position-specific scoring matrix (SB-PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB-PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA-based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi-scale pipeline can readily be applied to other protein-peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence-based prediction models is not available. Proteins 2016; 84:1390-1407. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A Specific Peptide with Calcium-Binding Capacity from Defatted Schizochytrium sp. Protein Hydrolysates and the Molecular Properties.

PubMed

Cai, Xixi; Yang, Qian; Lin, Jiaping; Fu, Nanyan; Wang, Shaoyun

2017-03-29

Marine microorganisms have been proposed as a new kind of protein source. Efforts are needed in order to transform the protein-rich biological wastes left after lipid extraction into value-added bio-products. Thus, the utilization of protein recovered from defatted Schizochytrium sp. by-products presents an opportunity. A specific peptide Tyr-Leu (YL) with calcium-binding capacity was purified from defatted Schizochytrium sp. protein hydrolysates through gel filtration chromatography and RP-HPLC. The calcium-binding activity of YL reached 126.34 ± 3.40 μg/mg. The calcium-binding mechanism was investigated through ultraviolet, fluorescence and infrared spectroscopy. The results showed that calcium ions could form dative bonds with carboxyl oxygen atoms and amino nitrogen atoms as well as the nitrogen and oxygen atoms of amide bonds. YL-Ca exhibited excellent thermal stability and solubility, which was beneficial for its absorption and transport in the basic intestinal tract of the human body. Moreover, the cellular uptake of calcium in Caco-2 cells showed that YL-Ca could enhance calcium uptake efficiency and protect calcium ions against precipitation caused by dietary inhibitors such as tannic acid, oxalate, phytate and metal ions. The findings indicate that the by-product of Schizochytrium sp. is a promising source for making peptide-calcium bio-products as algae-based functional supplements for human beings.

Strong Electrostatic Interactions Lead to Entropically Favorable Binding of Peptides to Charged Surfaces.

PubMed

Sprenger, K G; Pfaendtner, Jim

2016-06-07

Thermodynamic analyses can provide key insights into the origins of protein self-assembly on surfaces, protein function, and protein stability. However, obtaining quantitative measurements of thermodynamic observables from unbiased classical simulations of peptide or protein adsorption is challenging because of sampling limitations brought on by strong biomolecule/surface binding forces as well as time scale limitations. We used the parallel tempering metadynamics in the well-tempered ensemble (PTMetaD-WTE) enhanced sampling method to study the adsorption behavior and thermodynamics of several explicitly solvated model peptide adsorption systems, providing new molecular-level insight into the biomolecule adsorption process. Specifically studied were peptides LKα14 and LKβ15 and trpcage miniprotein adsorbing onto a charged, hydrophilic self-assembled monolayer surface functionalized with a carboxylic acid/carboxylate headgroup and a neutral, hydrophobic methyl-terminated self-assembled monolayer surface. Binding free energies were calculated as a function of temperature for each system and decomposed into their respective energetic and entropic contributions. We investigated how specific interfacial features such as peptide/surface electrostatic interactions and surface-bound ion content affect the thermodynamic landscape of adsorption and lead to differences in surface-bound conformations of the peptides. Results show that upon adsorption to the charged surface, configurational entropy gains of the released solvent molecules dominate the configurational entropy losses of the bound peptide. This behavior leads to an apparent increase in overall system entropy upon binding and therefore to the surprising and seemingly nonphysical result of an apparent increased binding free energy at elevated temperatures. Opposite effects and conclusions are found for the neutral surface. Additional simulations demonstrate that by adjusting the ionic strength of the solution

Evaluation of Phosphatidylserine-Binding Peptides Radiolabeled with Fluorine 18 for in vivo Imaging of Apoptosis

NASA Astrophysics Data System (ADS)

Kapty, Janice Sarah

We currently do not have a clinical method to directly assess apoptosis induced by cancer therapies. Phosphatidylserine (PS) is an attractive target for imaging apoptosis since it is on the exterior of the apoptotic cells and PS externalization is an early marker of apoptosis. PS-binding peptides are an attractive option for developing an imaging probe to detect apoptosis using positron emission tomography. In this study we evaluated binding characteristics of PS-binding peptides for ability to bind to PS, radiolabeled PS-binding peptides with fluorine-18, and performed in vitro and in vivo analysis of 18F radiolabeled PS-binding peptides including biodistribution analysis and dynamic PET imaging in a murine tumor model of apoptosis. Four peptides were evaluated for PS binding characteristics using a plate based assay system, a liposome mimic of cell membrane PS presentation, and a cell assay of apoptosis. The results indicate that all four peptides bind to PS and are specific to apoptotic cells. The widely used 18 F prosthetic group N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) and the recently developed N-[6-(4-[ 18F]fluorobenzylidene) aminooxyhexyl]maleimide ([18F]FBAM) were investigated for radiolabeling of two representative phosphatidylserine-binding peptides. The prosthetic groups were compared with respect to required reaction conditions for optimum labeling, radiolabeling yield and chemoselectivity. The N-terminus labeled product produced by reaction of [18F]SFB with binding peptide LIKKPF was produced in 18% radiochemical yield while no N-terminus labeled product could be isolated following [18F]SFB reaction with PDGLSR. When the peptides were modified by addition of a cysteine residue at the N-terminus they provided almost quantitative radiochemical yields with [18F]FBAM. Results indicate that for the peptides in this study, [18F]FBAM is a more useful prosthetic group compared to [18F]SFB due to its excellent chemo-selectivity and high radiochemical

Identification and characterization of a salivary-pellicle-binding peptide by phage display.

PubMed

Cukkemane, Nivedita; Bikker, Floris J; Nazmi, Kamran; Brand, Henk S; Veerman, Enno C I

2014-05-01

Dental biofilms are associated with oral diseases, making their control necessary. One way to control them is to prevent initial bacterial adherence to the salivary pellicle and thereby eventually decrease binding of late colonizing potential pathogens. The goal of this study was to generate a salivary-pellicle-binding peptide (SPBP) with antifouling activity towards primary colonizing bacteria. In order to achieve this goal we aimed to: (i) identify novel SPBPs by phage display; (ii) characterize the binding and antifouling properties of the selected SPBPs. A library of 2×10(9) phages displaying a random sequence of 12-mer peptides was used to identify peptides that bound selectively to the in vitro salivary pellicle. Three rounds of panning resulted in the selection of 10 pellicle-binding phages, each displaying a novel peptide sequence. The peptides were synthesized and their binding to the in vitro salivary pellicle was characterized in the presence and absence of calcium ions and Tween-20. The antifouling property of hydroxyapatite (HA) and saliva-coated HA discs treated with and without SPBPs were evaluated against Streptococcus gordonii. Ten unique SPBPs were identified using the phage display. One of these peptides, SPBP 10 (NSAAVRAYSPPS), exhibited significant binding to the in vitro salivary pellicle which was neither influenced by calcium ions, nor affected by up to 0.5% Tween-20. Its antifouling property against S. gordonii was significantly higher on the treated surfaces than on untreated surfaces. Use of the phage display library enabled us to find a specific SPBP with antifouling property towards S. gordonii. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization of particulate matter binding peptides screened from phage display.

PubMed

Liang Alvin, Aw Wei; Tanaka, Masayoshi; Okochi, Mina

2017-05-01

Particulate matter (PM), especially particulates with diameters of less than 2.5 μm, can penetrate the alveolar region and increase the risk of respiratory diseases. This has stimulated research efforts to develop detection methods so that counter measures can be taken. In this study, four PM binding peptides were obtained by phage display and binding characteristics of these peptides were investigated using the peptide array. The strongest binding peptide, WQDFGAVRSTRS, displayed a binding property, measured in terms of spot intensity, 11.4 times higher than that of the negative control, AAAAA. Inductively coupled plasma mass spectrometry (ICPMS) analysis of the transition metal compounds in the PM bound to the peptide spots was performed, and two peptides showed higher binding towards Cu and Zn compounds in PM. These results suggest that the screened peptides could serve as an indicator of transition metal compounds, which are related to adverse health effects, contained in PM. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Determinants of BH3 binding specificity for Mcl-1 vs. Bcl-xL

PubMed Central

Dutta, Sanjib; Gullá, Stefano; Chen, T. Scott; Fire, Emiko; Grant, Robert A.; Keating, Amy E.

2010-01-01

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Pro-survival members of the family interact with pro-apoptotic BH3-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the alpha-helical BH3 region of the pro-apoptotic proteins to a conserved hydrophobic groove on the pro-survival proteins. Native BH3-only proteins exhibit selectivity in binding pro-survival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work we used two complementary techniques, yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis, to elucidate specificity determinants for binding to Bcl-xL vs. Mcl-1, two prominent pro-survival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-xL selectively, or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1 selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-xL, Bcl-2, Bcl-w and Bfl-1, whereas Bcl-xL selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 vs. Bcl-xL binders. PMID:20363230

Identification of lanthanum-specific peptides for future recycling of rare earth elements from compact fluorescent lamps.

PubMed

Lederer, Franziska L; Curtis, Susan B; Bachmann, Stefanie; Dunbar, W Scott; MacGillivray, Ross T A

2017-05-01

As components of electronic scrap, rare earth minerals are an interesting but little used source of raw materials that are highly important for the recycling industry. Currently, there exists no cost-efficient technology to separate rare earth minerals from an electronic scrap mixture. In this study, phage surface display has been used as a key method to develop peptides with high specificity for particular inorganic targets in electronic scrap. Lanthanum phosphate doped with cerium and terbium as part of the fluorescent phosphors of spent compact fluorescent lamps (CFL) was used as a target material of economic interest to test the suitability of the phage display method to the separation of rare earth minerals. One random pVIII phage library was screened for peptide sequences that bind specifically to the fluorescent phosphor LaPO 4 :Ce 3+ ,Tb 3+ (LAP). The library contained at least 100 binding pVIII peptides per phage particle with a diversity of 1 × 10 9 different phage per library. After three rounds of enrichment, a phage clone containing the surface peptide loop RCQYPLCS was found to bind specifically to LAP. Specificity and affinity of the identified phage bound peptide was confirmed by using binding and competition assays, immunofluorescence assays, and zeta potential measurements. Binding and immunofluorescence assays identified the peptide's affinity for the fluorescent phosphor components CAT (CeMgAl 11 O 19 :Tb 3+ ) and BAM (BaMgAl 10 O 17 :Eu 2+ ). No affinity was found for other fluorescent phosphor components such as YOX (Y 2 O 3 :Eu 3+ ). The binding specificity of the RCQYPLCS peptide loop was improved 3-51-fold by using alanine scanning mutagenesis. The identification of peptides with high specificity and affinity for special components in the fluorescent phosphor in CFLs provides a potentially new strategic approach to rare earth recycling. Biotechnol. Bioeng. 2017;114: 1016-1024. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals

Novel thrombopoietin mimetic peptides bind c-Mpl receptor: Synthesis, biological evaluation and molecular modeling.

PubMed

Liu, Yaquan; Tian, Fang; Zhi, Dejuan; Wang, Haiqing; Zhao, Chunyan; Li, Hongyu

2017-02-01

Thrombopoietin (TPO) acts in promoting the proliferation of hematopoietic stem cells and by initiating specific maturation events in megakaryocytes. Now, TPO-mimetic peptides with amino acid sequences unrelated to TPO are of considerable pharmaceutical interest. In the present paper, four new TPO mimetic peptides that bind and activate c-Mpl receptor have been identified, synthesized and tested by Dual-Luciferase reporter gene assay for biological activities. The molecular modeling research was also approached to understand key molecular mechanisms and structural features responsible for peptide binding with c-Mpl receptor. The results presented that three of four mimetic peptides showed significant activities. In addition, the molecular modeling approaches proved hydrophobic interactions were the driven positive forces for binding behavior between peptides and c-Mpl receptor. TPO peptide residues in P7, P13 and P7' positions were identified by the analysis of hydrogen bonds and energy decompositions as the key ones for benefiting better biological activities. Our data suggested the synthesized peptides have considerable potential for the future development of stable and highly active TPO mimetic peptides. Copyright © 2016 Elsevier Ltd. All rights reserved.

Solid-Binding Peptides in Biomedicine.

PubMed

Care, Andrew; Bergquist, Peter L; Sunna, Anwar

2017-01-01

Some peptides are able to bind to inorganic materials such as silica and gold. Over the past decade, Solid-binding peptides (SBPs) have been used increasingly as molecular building blocks in nanobiotechnology. These peptides show selectivity and bind with high affinity to a diverse range of inorganic surfaces e.g. metals, metal oxides, metal compounds, magnetic materials, semiconductors, carbon materials, polymers and minerals. They can be used in applications such as protein purification and synthesis, assembly and the functionalization of nanomaterials. They offer simple and versatile bioconjugation methods that can increase biocompatibility and also direct the immobilization and orientation of nanoscale entities onto solid supports without impeding their functionality. SBPs have been employed in numerous nanobiotechnological applications such as the controlled synthesis of nanomaterials and nanostructures, formation of hybrid biomaterials, immobilization of functional proteins and improved nanomaterial biocompatibility. With advances in nanotechnology, a multitude of novel nanomaterials have been designed and synthesized for diagnostic and therapeutic applications. New approaches have been developed recently to exert a greater control over bioconjugation and eventually, over the optimal and functional display of biomolecules on the surfaces of many types of solid materials. In this chapter we describe SBPs and highlight some selected examples of their potential applications in biomedicine.

Secondary structure propensity and chirality of the amyloidophilic peptide p5 and its analogues impacts ligand binding - In vitro characterization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wall, Jonathan S.; Williams, Angela; Wooliver, Craig

Here, polybasic helical peptides, such as peptide p5, bind human amyloid extracts and synthetic amyloid fibrils. When radio labeled, peptide p5 has been shown to specifically bind amyloid in vivo thereby allowing imaging of the disease. Structural requirements for heparin and amyloid binding have been studied using analogues of p5 that modify helicity and chirality.

Secondary structure propensity and chirality of the amyloidophilic peptide p5 and its analogues impacts ligand binding - In vitro characterization

DOE PAGES

Wall, Jonathan S.; Williams, Angela; Wooliver, Craig; ...

2016-08-11

Here, polybasic helical peptides, such as peptide p5, bind human amyloid extracts and synthetic amyloid fibrils. When radio labeled, peptide p5 has been shown to specifically bind amyloid in vivo thereby allowing imaging of the disease. Structural requirements for heparin and amyloid binding have been studied using analogues of p5 that modify helicity and chirality.

Rapid discovery of peptide capture candidates with demonstrated specificity for structurally similar toxins

NASA Astrophysics Data System (ADS)

Sarkes, Deborah A.; Hurley, Margaret M.; Coppock, Matthew B.; Farrell, Mikella E.; Pellegrino, Paul M.; Stratis-Cullum, Dimitra N.

2016-05-01

Peptides have emerged as viable alternatives to antibodies for molecular-based sensing due to their similarity in recognition ability despite their relative structural simplicity. Various methods for peptide capture reagent discovery exist, including phage display, yeast display, and bacterial display. One of the primary advantages of peptide discovery by bacterial display technology is the speed to candidate peptide capture agent, due to both rapid growth of bacteria and direct utilization of the sorted cells displaying each individual peptide for the subsequent round of biopanning. We have previously isolated peptide affinity reagents towards protective antigen of Bacillus anthracis using a commercially available automated magnetic sorting platform with improved enrichment as compared to manual magnetic sorting. In this work, we focus on adapting our automated biopanning method to a more challenging sort, to demonstrate the specificity possible with peptide capture agents. This was achieved using non-toxic, recombinant variants of ricin and abrin, RiVax and abrax, respectively, which are structurally similar Type II ribosomal inactivating proteins with significant sequence homology. After only two rounds of biopanning, enrichment of peptide capture candidates binding abrax but not RiVax was achieved as demonstrated by Fluorescence Activated Cell Sorting (FACS) studies. Further sorting optimization included negative sorting against RiVax, proper selection of autoMACS programs for specific sorting rounds, and using freshly made buffer and freshly thawed protein target for each round of biopanning for continued enrichment over all four rounds. Most of the resulting candidates from biopanning for abrax binding peptides were able to bind abrax but not RiVax, demonstrating that short peptide sequences can be highly specific even at this early discovery stage.

A Fluorescent Protein Scaffold for Presenting Structurally Constrained Peptides Provides an Effective Screening System to Identify High Affinity Target-Binding Peptides

PubMed Central

Kadonosono, Tetsuya; Yabe, Etsuri; Furuta, Tadaomi; Yamano, Akihiro; Tsubaki, Takuya; Sekine, Takuya; Kuchimaru, Takahiro; Sakurai, Minoru; Kizaka-Kondoh, Shinae

2014-01-01

Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131–L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2)-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides. PMID:25084350

Biomaterials functionalization using a novel peptide that selectively binds to a conducting polymer

NASA Astrophysics Data System (ADS)

Sanghvi, Archit B.; Miller, Kiley P.-H.; Belcher, Angela M.; Schmidt, Christine E.

2005-06-01

The goal in biomaterial surface modification is to retain a material's bulk properties while modifying only its surface to possess desired recognition and specificity. Here we develop a unique strategy for surface functionalization of an electrically conductive polymer, chlorine-doped polypyrrole (PPyCl), which has been widely researched for various electronic and biomedical applications. An M13 bacteriophage library was used to screen 109 different 12-mer peptide inserts against PPyCl. A binding phage (ϕT59) was isolated, and its binding stability and specificity to PPyCl was assessed using fluorescence microscopy and titer count analysis. The relative binding strength and mechanism of the corresponding 12-mer peptide and its variants was studied using atomic force microscopy and fluorescamine assays. Further, the T59 peptide was joined to a cell adhesive sequence and used to promote cell attachment on PPyCl. This strategy can be extended to immobilize a variety of molecules to PPyCl for numerous applications. In addition, phage display can be applied to other polymers to develop bioactive materials without altering their bulk properties.

MHC2NNZ: A novel peptide binding prediction approach for HLA DQ molecules

NASA Astrophysics Data System (ADS)

Xie, Jiang; Zeng, Xu; Lu, Dongfang; Liu, Zhixiang; Wang, Jiao

2017-07-01

The major histocompatibility complex class II (MHC-II) molecule plays a crucial role in immunology. Computational prediction of MHC-II binding peptides can help researchers understand the mechanism of immune systems and design vaccines. Most of the prediction algorithms for MHC-II to date have made large efforts in human leukocyte antigen (HLA, the name of MHC in Human) molecules encoded in the DR locus. However, HLA DQ molecules are equally important and have only been made less progress because it is more difficult to handle them experimentally. In this study, we propose an artificial neural network-based approach called MHC2NNZ to predict peptides binding to HLA DQ molecules. Unlike previous artificial neural network-based methods, MHC2NNZ not only considers sequence similarity features but also captures the chemical and physical properties, and a novel method incorporating these properties is proposed to represent peptide flanking regions (PFR). Furthermore, MHC2NNZ improves the prediction accuracy by combining with amino acid preference at more specific positions of the peptides binding core. By evaluating on 3549 peptides binding to six most frequent HLA DQ molecules, MHC2NNZ is demonstrated to outperform other state-of-the-art MHC-II prediction methods.

Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

PubMed

Hou, Peili; Zhao, Guimin; He, Chengqiang; Wang, Hongmei; He, Hongbin

2018-01-04

The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G 1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G 1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G 1 protein with high-affinity and inhibit BEFV replication. The purified BEFV G 1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G 1 was assayed by ELISA. Then the roles of specific G 1 -binding peptides in the context of BEFV infection were analyzed. The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G 1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. Two antiviral peptide ligands binding to bovine ephemeral fever virus G 1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.

Connecting Effective Immune Response, Fluorescent Granzyme B-like Peptide, Specific Peptide Binding Patterns, Patients with Cancer and Viral Infection, in Remission, Clinical Significance, and Liquid Biopsy.

PubMed

Lo, Wai Chun Jennifer; Luther, Donald Gene

2016-11-01

Functional cytotoxic-T-lymphocytes (CTL) with granzyme B play an important role in an effective immune response to tumor growth and infection progression. Tumor cells and platelets in peripheral whole blood smears of cancer patients have shown the presence of innate binding targets for GP1R, a fluorescent synthetic Granzyme B-like peptide. It is not known if similar GP1R-binding targets and specific binding patterns are detectable in peripheral blood of patients with viral infection. It is also not known if a specific binding pattern may be associated with an effective immune response to indicate a favorable prognosis. We reviewed the GP1R-binding patterns in the peripheral blood smears of 5 patients in remission at the time of sampling (3 with cancer and 2 with flu-like symptoms) and a negative control. We show with fluoroscopic images that there are: 1) fluorescent GP1R-binding targets mostly in the cytoplasmic areas of nucleated cells in patients with breast and lung cancer who have longer survival, 2) intense fluorescent deposits mostly in the nuclear areas of segmented neutrophils in patients recovered from severe to mild flu-like symptoms, 3) discernible fluorescent deposits in the cytoplasmic areas of small lymphocyte-like elements and overall intense fluorescent stain in large cells in the patient with advanced pancreatic cancer who had shorter survival, 4) GP1R-binding targets in numerous platelet-like elements in all 5 patients. The control sample did not show similar binding patterns. The potential association between specific GP1R-binding patterns in peripheral blood samples and prognostic significance, and its use as liquid biopsy are discussed.

Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-x[subscript L

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dutta, Sanjib; Gullá, Stefano; Chen, T. Scott

2010-06-25

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the {alpha}-helical BH3 region of the proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the designmore » of new classes of selective inhibitors to serve as reagents or therapeutics. In this work, we used two complementary techniques - yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis - to elucidate specificity determinants for binding to Bcl-x{sub L} versus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x{sub L} selectively or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1-selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-x{sub L}, Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x{sub L}-selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 versus Bcl-x{sub L} binders.« less

Discovery of 12-mer peptides that bind to wood lignin

PubMed Central

Yamaguchi, Asako; Isozaki, Katsuhiro; Nakamura, Masaharu; Takaya, Hikaru; Watanabe, Takashi

2016-01-01

Lignin, an abundant terrestrial polymer, is the only large-volume renewable feedstock composed of an aromatic skeleton. Lignin has been used mostly as an energy source during paper production; however, recent interest in replacing fossil fuels with renewable resources has highlighted its potential value in providing aromatic chemicals. Highly selective degradation of lignin is pivotal for industrial production of paper, biofuels, chemicals, and materials. However, few studies have examined natural and synthetic molecular components recognizing the heterogeneous aromatic polymer. Here, we report the first identification of lignin-binding peptides possessing characteristic sequences using a phage display technique. The consensus sequence HFPSP was found in several lignin-binding peptides, and the outer amino acid sequence affected the binding affinity of the peptides. Substitution of phenylalanine7 with Ile in the lignin-binding peptide C416 (HFPSPIFQRHSH) decreased the affinity of the peptide for softwood lignin without changing its affinity for hardwood lignin, indicating that C416 recognised structural differences between the lignins. Circular dichroism spectroscopy demonstrated that this peptide adopted a highly flexible random coil structure, allowing key residues to be appropriately arranged in relation to the binding site in lignin. These results provide a useful platform for designing synthetic and biological catalysts selectively bind to lignin. PMID:26903196

Interaction of Nevirapine with the Peptide Binding Groove of HLA-DRB1*01:01 and Its Effect on the Conformation of HLA-Peptide Complex.

PubMed

Hirasawa, Makoto; Hagihara, Katsunobu; Abe, Koji; Ando, Osamu; Hirayama, Noriaki

2018-06-04

Human leukocyte antigen (HLA)-DRB1*01:01 has been shown to be involved in nevirapine-induced hepatic hypersensitivity reactions. In the present study, in silico docking simulations and molecular dynamics simulations were performed to predict the interaction mode of nevirapine with the peptide binding groove of HLA-DRB1*01:01 and its possible effect on the position and orientation of the ligand peptide derived from hemagglutinin (HA). In silico analyses suggested that nevirapine interacts with HLA-DRB1*01:01 around the P4 pocket within the peptide binding groove and the HA peptide stably binds on top of nevirapine at the groove. The analyses also showed that binding of nevirapine at the groove will significantly change the inter-helical distances of the groove. An in vitro competitive assay showed that nevirapine (1000 μM) increases the binding of the HA peptide to HLA-DRB1*01:01 in an allele-specific manner. These results indicate that nevirapine might interact directly with the P4 pocket and modifies its structure, which could change the orientation of loaded peptides and the conformation of HLA-DRB1*01:01; these changes could be distinctively recognized by T-cell receptors. Through this molecular mechanism, nevirapine might stimulate the immune system, resulting in hepatic hypersensitivity reactions.

Stiffening of flexible SUMO1 protein upon peptide-binding: Analysis with anisotropic network model.

PubMed

Sarkar, Ranja

2018-01-01

SUMO (small ubiquitin-like modifier) proteins interact with a large number of target proteins via a key regulatory event called sumoylation that encompasses activation, conjugation and ligation of SUMO proteins through specific E1, E2, and E3-type enzymes respectively. Single-molecule atomic force microscopic (AFM) experiments performed to unravel bound SUMO1 along its NC termini direction reveal that E3-ligases (in the form of small peptides) increase mechanical stability (along the axis) of the flexible protein upon binding. The experimental results are expected to correlate with the intrinsic flexibility of bound SUMO1 protein in the native state i.e., the bound conformation of SUMO1 without the binding peptide. The native protein flexibility/stiffness can be measured as a spring constant by normal mode analysis. In the present study, protein normal modes are computed from the protein structural data (as input from protein databank) via a simple anisotropic network model (ANM). ANM is computationally inexpensive and hence, can be explored to investigate and compare the native conformational dynamics of unbound and bound (without the binding partner) structures, if the corresponding structural data (NMR/X-ray) are available. The paper illustrates that SUMO1 stiffens (native flexibility decreases) along the NC termini (end-to-end) direction of the protein upon binding to small peptides; however, the degree of stiffening is peptide sequence-specific. The theoretical results are demonstrated for NMR structures of unbound SUMO1 and that bound to two peptides having short amino acid motifs and of similar size, one being an M-IR2 peptide derived from RanBP2 protein and the other one derived from PIASX protein. The peptide derived from PIASX stiffens SUMO1 remarkably which is evident from an atomic-level normal mode analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

PSSMHCpan: a novel PSSM-based software for predicting class I peptide-HLA binding affinity

PubMed Central

Liu, Geng; Li, Dongli; Li, Zhang; Qiu, Si; Li, Wenhui; Chao, Cheng-chi; Yang, Naibo; Li, Handong; Cheng, Zhen; Song, Xin; Cheng, Le; Zhang, Xiuqing; Wang, Jian; Yang, Huanming

2017-01-01

Abstract Predicting peptide binding affinity with human leukocyte antigen (HLA) is a crucial step in developing powerful antitumor vaccine for cancer immunotherapy. Currently available methods work quite well in predicting peptide binding affinity with HLA alleles such as HLA-A*0201, HLA-A*0101, and HLA-B*0702 in terms of sensitivity and specificity. However, quite a few types of HLA alleles that are present in the majority of human populations including HLA-A*0202, HLA-A*0203, HLA-A*6802, HLA-B*5101, HLA-B*5301, HLA-B*5401, and HLA-B*5701 still cannot be predicted with satisfactory accuracy using currently available methods. Furthermore, currently the most popularly used methods for predicting peptide binding affinity are inefficient in identifying neoantigens from a large quantity of whole genome and transcriptome sequencing data. Here we present a Position Specific Scoring Matrix (PSSM)-based software called PSSMHCpan to accurately and efficiently predict peptide binding affinity with a broad coverage of HLA class I alleles. We evaluated the performance of PSSMHCpan by analyzing 10-fold cross-validation on a training database containing 87 HLA alleles and obtained an average area under receiver operating characteristic curve (AUC) of 0.94 and accuracy (ACC) of 0.85. In an independent dataset (Peptide Database of Cancer Immunity) evaluation, PSSMHCpan is substantially better than the popularly used NetMHC-4.0, NetMHCpan-3.0, PickPocket, Nebula, and SMM with a sensitivity of 0.90, as compared to 0.74, 0.81, 0.77, 0.24, and 0.79. In addition, PSSMHCpan is more than 197 times faster than NetMHC-4.0, NetMHCpan-3.0, PickPocket, sNebula, and SMM when predicting neoantigens from 661 263 peptides from a breast tumor sample. Finally, we built a neoantigen prediction pipeline and identified 117 017 neoantigens from 467 cancer samples of various cancers from TCGA. PSSMHCpan is superior to the currently available methods in predicting peptide binding affinity with a

Exploring the active site binding specificity of kallikrein-related peptidase 5 (KLK5) guides the design of new peptide substrates and inhibitors.

PubMed

de Veer, Simon J; Swedberg, Joakim E; Brattsand, Maria; Clements, Judith A; Harris, Jonathan M

2016-12-01

Kallikrein-related peptidase 5 (KLK5) is a promising therapeutic target in several skin diseases, including Netherton syndrome, and is emerging as a potential target in various cancers. In this study, we used a sparse matrix library of 125 individually synthesized peptide substrates to characterize the binding specificity of KLK5. The sequences most favored by KLK5 were GRSR, YRSR and GRNR, and we identified sequence-specific interactions involving the peptide N-terminus by analyzing kinetic constants (kcat and KM) and performing molecular dynamics simulations. KLK5 inhibitors were subsequently engineered by substituting substrate sequences into the binding loop (P1, P2 and P4 residues) of sunflower trypsin inhibitor-1 (SFTI-1). These inhibitors were effective against KLK5 but showed limited selectivity, and performing a further substitution at P2' led to the design of a new variant that displayed improved activity against KLK5 (Ki=4.2±0.2 nm), weak activity against KLK7 and 12-fold selectivity over KLK14. Collectively, these findings provide new insight into the design of highly favored binding sequences for KLK5 and reveal several opportunities for modulating inhibitor selectivity over closely related proteases that will be useful for future studies aiming to develop therapeutic molecules targeting KLK5.

Scrutinizing MHC-I binding peptides and their limits of variation.

PubMed

Koch, Christian P; Perna, Anna M; Pillong, Max; Todoroff, Nickolay K; Wrede, Paul; Folkers, Gerd; Hiss, Jan A; Schneider, Gisbert

2013-01-01

Designed peptides that bind to major histocompatibility protein I (MHC-I) allomorphs bear the promise of representing epitopes that stimulate a desired immune response. A rigorous bioinformatical exploration of sequence patterns hidden in peptides that bind to the mouse MHC-I allomorph H-2K(b) is presented. We exemplify and validate these motif findings by systematically dissecting the epitope SIINFEKL and analyzing the resulting fragments for their binding potential to H-2K(b) in a thermal denaturation assay. The results demonstrate that only fragments exclusively retaining the carboxy- or amino-terminus of the reference peptide exhibit significant binding potential, with the N-terminal pentapeptide SIINF as shortest ligand. This study demonstrates that sophisticated machine-learning algorithms excel at extracting fine-grained patterns from peptide sequence data and predicting MHC-I binding peptides, thereby considerably extending existing linear prediction models and providing a fresh view on the computer-based molecular design of future synthetic vaccines. The server for prediction is available at http://modlab-cadd.ethz.ch (SLiDER tool, MHC-I version 2012).

HLA Class I Binding 9mer Peptides from Influenza A Virus Induce CD4+ T Cell Responses

PubMed Central

Wang, Mingjun; Larsen, Mette V.; Nielsen, Morten; Harndahl, Mikkel; Justesen, Sune; Dziegiel, Morten H.; Buus, Søren; Tang, Sheila T.; Lund, Ole; Claesson, Mogens H.

2010-01-01

Background Identification of human leukocyte antigen class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes from influenza virus is of importance for the development of new effective peptide-based vaccines. Methodology/Principal Findings In the present work, bioinformatics was used to predict 9mer peptides derived from available influenza A viral proteins with binding affinity for at least one of the 12 HLA-I supertypes. The predicted peptides were then selected in a way that ensured maximal coverage of the available influenza A strains. One hundred and thirty one peptides were synthesized and their binding affinities for the HLA-I supertypes were measured in a biochemical assay. Influenza-specific T cell responses towards the peptides were quantified using IFNγ ELISPOT assays with peripheral blood mononuclear cells (PBMC) from adult healthy HLA-I typed donors as responder cells. Of the 131 peptides, 21 were found to induce T cell responses in 19 donors. In the ELISPOT assay, five peptides induced responses that could be totally blocked by the pan-specific anti-HLA-I antibody W6/32, whereas 15 peptides induced responses that could be completely blocked in the presence of the pan-specific anti-HLA class II (HLA-II) antibody IVA12. Blocking of HLA-II subtype reactivity revealed that 8 and 6 peptide responses were blocked by anti-HLA-DR and -DP antibodies, respectively. Peptide reactivity of PBMC depleted of CD4+ or CD8+ T cells prior to the ELISPOT culture revealed that effectors are either CD4+ (the majority of reactivities) or CD8+ T cells, never a mixture of these subsets. Three of the peptides, recognized by CD4+ T cells showed binding to recombinant DRA1*0101/DRB1*0401 or DRA1*0101/DRB5*0101 molecules in a recently developed biochemical assay. Conclusions/Significance HLA-I binding 9mer influenza virus-derived peptides induce in many cases CD4+ T cell responses restricted by HLA-II molecules. PMID:20479886

Cardiac distribution of the binding sites for natriuretic peptides in vertebrates.

PubMed

Cerra, M C

1994-12-01

Natriuretic peptides are hormones that play an important role in the cardiovascular control of mammalian and non-mammalian vertebrates. They have been classified into four groups. Of these, ANP (atrial natriuretic peptide), BNP (brain atriuretic peptides), CNP (C-type natriuretic peptide) are detected in cardiac and non cardiac tissues of all vertebrates; while VNP (ventricular natriuretic peptide) has been isolated only from the fish ventricle. All peptides have shown a high degree of sequence homology. The expression of the three principal types of natriuretic peptide (ANP, BNP and CNP) in cardiac tissues is developmentally and functionally regulated in a highly tissue-specific manner. Three types of natriuretic peptide receptors have been identified in numerous target tissues. Two receptors are transmembrane guanylyl cyclases (ANPR-A and ANPR-B) that mediate biological effects of natriuretic peptides; the third one (ANPR-C) has no guanylyl cyclase and is called "clearance receptor." The presence of natriuretic peptide binding sites in the heart suggests new aspects of paracrine control of cardiac function. A relevant localization of natriuretic peptide receptors was found in those cardiac regions particularly suitable for monitoring blood volume and pressure oscillations such as the inflow tract and the outflow tract. For example, in birds (quail) the highest levels of natriuretic peptide receptors were detected in the inflow tract represented by the vena cava. In both fish and birds, the outflow chamber, the bulbus cordis, had a high number of natriuretic peptide binding sites. In mammals, a remarkable concentration of natriuretic peptide receptors was also observed in the coronary vessels. This zoning of cardiac natriuretic peptide receptors indicates an intracardiac action of the hormones and adds a humoral dimension to the morphofunctional design of the vertebrate heart.

Characterization of binding specificities of Bovine Leucocyte class I molecules: Impacts for rational epitope discovery

USDA-ARS?s Scientific Manuscript database

The binding of peptides to classical major histocompatibility complex (MHC) class-I proteins is the single most selective step in antigen presentation. However, the peptide binding specificity of cattle MHC (bovine leucocyte antigen, BoLA) class I (BoLA-I) molecules remains poorly characterized. Her...

Size-controlled synthesis of Pd nanocrystals using a specific multifunctional peptide

NASA Astrophysics Data System (ADS)

Chiu, Chin-Yi; Li, Yujing; Huang, Yu

2010-06-01

Here we report a peptide-mediated synthesis of Pd NCs in aqueous solution with controllable size in the sub-10 nanometre regime. The specific multifunctional peptide Q7 selected using the phage display technique can bind to the Pd NC surface and act as a stabilizer to mediate Pd crystal nucleation and growth. At the nucleation stage, Q7 bound to and helped stabilize the different-sized small Pd NC nuclei achieved using different concentrations of the external reducing agent, NaBH4. At the growth stage, Q7 played the dual role of binding to and reducing the precursor onto the existing nuclei, which led to the further controllable growth of the Pd NCs. By using the variable sizes of nuclei as seeds, and by introducing different amounts of precursors Pd NCs with tunable sizes from 2.6 to 6.6 nm were achieved with good size distribution.Here we report a peptide-mediated synthesis of Pd NCs in aqueous solution with controllable size in the sub-10 nanometre regime. The specific multifunctional peptide Q7 selected using the phage display technique can bind to the Pd NC surface and act as a stabilizer to mediate Pd crystal nucleation and growth. At the nucleation stage, Q7 bound to and helped stabilize the different-sized small Pd NC nuclei achieved using different concentrations of the external reducing agent, NaBH4. At the growth stage, Q7 played the dual role of binding to and reducing the precursor onto the existing nuclei, which led to the further controllable growth of the Pd NCs. By using the variable sizes of nuclei as seeds, and by introducing different amounts of precursors Pd NCs with tunable sizes from 2.6 to 6.6 nm were achieved with good size distribution. Electronic Supplementary Information (ESI) available. Experimental details for peptide selection, peptide synthesis and Pd NCs synthesis; Q7 peptide sequence molecular structure and characterization; TEM images of Pd NCs. See DOI: 10.1039/c0nr00194e/

Peptide ligands specific to the oxidized form of escherichia coli thioredoxin.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scholle, M. D.; Banach, B. S.; Hamdan, S. M.

Thioredoxin (Trx) is a highly conserved redox protein involved in several essential cellular processes. In this study, our goal was to isolate peptide ligands to Escherichia coli Trx that mimic protein-protein interactions, specifically the T7 polymerase-Trx interaction. To do this, we subjected Trx to affinity selection against a panel of linear and cysteine-constrained peptides using M13 phage display. A novel cyclized conserved peptide sequence, with a motif of C(D/N/S/T/G)D(S/T)-hydrophobic-C-X-hydrophobic-P, was isolated to Trx. These peptides bound specifically to the E. coli Trx when compared to the human and spirulina homologs. An alanine substitution of the active site cysteines (CGPC) resultedmore » in a significant loss of peptide binding affinity to the Cys-32 mutant. The peptides were also characterized in the context of Trx's role as a processivity factor of the T7 DNA polymerase (gp5). As the interaction between gp5 and Trx normally takes place under reducing conditions, which might interfere with the conformation of the disulfide-bridged peptides, we made use of a 22 residue deletion mutant of gp5 in the thioredoxin binding domain (gp5{Delta}22) that bypassed the requirements of reducing conditions to interact with Trx. A competition study revealed that the peptide selectively inhibits the interaction of gp5{Delta}22 with Trx, under oxidizing conditions, with an IC50 of {approx} 10 {micro}M.« less

Biodiscovery of Aluminum Binding Peptides

DTIC Science & Technology

2013-08-01

et al., "Biomimetic synthesis and patterning of silver nanoparticles ," Nat. Mater. 1(3), 169-172 (2002). [5] Van Dorst, B., et al., "Phage display...34Sequestration of zinc oxide by fimbrial designer chelators," Appl. Environ. Microbiol. 66(1), 10-14 (2000). [26] Hnilova, M., et al., "Peptide-directed co...biomaterial synthesis . Peptides have been developed that bind to a variety of inorganic materials, including metals1-6, oxides7, 8, alloys9, metal salts10

Computational Studies of Difference in Binding Modes of Peptide and Non-Peptide Inhibitors to MDM2/MDMX Based on Molecular Dynamics Simulations

PubMed Central

Chen, Jianzhong; Zhang, Dinglin; Zhang, Yuxin; Li, Guohui

2012-01-01

Inhibition of p53-MDM2/MDMX interaction is considered to be a promising strategy for anticancer drug design to activate wild-type p53 in tumors. We carry out molecular dynamics (MD) simulations to study the binding mechanisms of peptide and non-peptide inhibitors to MDM2/MDMX. The rank of binding free energies calculated by molecular mechanics generalized Born surface area (MM-GBSA) method agrees with one of the experimental values. The results suggest that van der Waals energy drives two kinds of inhibitors to MDM2/MDMX. We also find that the peptide inhibitors can produce more interaction contacts with MDM2/MDMX than the non-peptide inhibitors. Binding mode predictions based on the inhibitor-residue interactions show that the π–π, CH–π and CH–CH interactions dominated by shape complimentarity, govern the binding of the inhibitors in the hydrophobic cleft of MDM2/MDMX. Our studies confirm the residue Tyr99 in MDMX can generate a steric clash with the inhibitors due to energy and structure. This finding may theoretically provide help to develop potent dual-specific or MDMX inhibitors. PMID:22408446

Staphylococcal surface display of metal-binding polyhistidyl peptides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Samuelson, P.; Wernerus, H.; Svedberg, M.

2000-03-01

Recombinant Staphylococcus xylosus and Staphylococcus carnosus strains were generated with surface-exposed chimeric proteins containing polyhistidyl peptides designed for binding to divalent metal ions. Surface accessibility of the chimeric surface proteins was demonstrated and the chimeric surface proteins were found to be functional in terms of metal binding, since the recombinant staphylococcal cells were shown to have gained Ni{sup 2+}- and Cd{sup 2+}-binding capacity, suggesting that such bacteria could find use in bioremediation of heavy metals. This is, to their knowledge, the first time that recombinant, surface-exposed metal-binding peptides have been expressed on gram-positive bacteria. Potential environmental or biosensor applications formore » such recombinant staphylococci as biosorbents are discussed.« less

The hydrophobic region of the DmsA twin-arginine leader peptide determines specificity with chaperone DmsD.

PubMed

Winstone, Tara M L; Tran, Vy A; Turner, Raymond J

2013-10-29

The system specific chaperone DmsD plays a role in the maturation of the catalytic subunit of dimethyl sulfoxide (DMSO) reductase, DmsA. Pre-DmsA contains a 45-amino acid twin-arginine leader peptide that is important for targeting and translocation of folded and cofactor-loaded DmsA by the twin-arginine translocase. DmsD has previously been shown to interact with the complete twin-arginine leader peptide of DmsA. In this study, isothermal titration calorimetry was used to investigate the thermodynamics of binding between synthetic peptides composed of different portions of the DmsA leader peptide and DmsD. Only those peptides that included the complete and contiguous hydrophobic region of the DmsA leader sequence were able to bind DmsD with a 1:1 stoichiometry. Each of the peptides that were able to bind DmsD also showed some α-helical structure as indicated by circular dichroism spectroscopy. Differential scanning calorimetry revealed that DmsD gained very little thermal stability upon binding any of the DmsA leader peptides tested. Together, these results suggest that a portion of the hydrophobic region of the DmsA leader peptide determines the specificity of binding and may produce helical properties upon binding to DmsD. Overall, this study demonstrates that the recognition of the DmsA twin-arginine leader sequence by the DmsD chaperone shows unexpected rules and confirms further that the biochemistry of the interaction of the chaperone with their leaders demonstrates differences in their molecular interactions.

Zn(II) and Hg(II) binding to a designed peptide that accommodates different coordination geometries.

PubMed

Szunyogh, Dániel; Gyurcsik, Béla; Larsen, Flemming H; Stachura, Monika; Thulstrup, Peter W; Hemmingsen, Lars; Jancsó, Attila

2015-07-28

Designed metal ion binding peptides offer a variety of applications in both basic science as model systems of more complex metalloproteins, and in biotechnology, e.g. in bioremediation of toxic metal ions, biomining or as artificial enzymes. In this work a peptide (HS: Ac-SCHGDQGSDCSI-NH2) has been specifically designed for binding of both Zn(II) and Hg(II), i.e. metal ions with different preferences in terms of coordination number, coordination geometry, and to some extent ligand composition. It is demonstrated that HS accommodates both metal ions, and the first coordination sphere, metal ion exchange between peptides, and speciation are characterized as a function of pH using UV-absorption-, synchrotron radiation CD-, (1)H-NMR-, and PAC-spectroscopy as well as potentiometry. Hg(II) binds to the peptide with very high affinity in a {HgS2} coordination geometry, bringing together the two cysteinates close to each end of the peptide in a loop structure. Despite the high affinity, Hg(II) is kinetically labile, exchanging between peptides on the subsecond timescale, as indicated by line broadening in (1)H-NMR. The Zn(II)-HS system displays more complex speciation, involving monomeric species with coordinating cysteinates, histidine, and a solvent water molecule, as well as HS-Zn(II)-HS complexes. In summary, the HS peptide displays conformational flexibility, contains many typical metal ion binding groups, and is able to accommodate metal ions with different structural and ligand preferences with high affinity. As such, the HS peptide may be a scaffold offering binding of a variety of metal ions, and potentially serve for metal ion sequestration in biotechnological applications.

Why Congo red binding is specific for amyloid proteins - model studies and a computer analysis approach.

PubMed

Roterman, I; KrUl, M; Nowak, M; Konieczny, L; Rybarska, J; Stopa, B; Piekarska, B; Zemanek, G

2001-01-01

The complexing of Congo red in two different ligand forms - unimolecular and supramolecular (seven molecules in a micelle) - with eight deca-peptides organized in a b-sheet was tested by computational analysis to identify its dye-binding preferences. Polyphenylananine and polylysine peptides were selected to represent the specific side chain interactions expected to ensure particularly the stabilization of the dye-protein complex. Polyalanine was used to verify the participation of non-specific backbone-derived interactions. The initial complexes for calculation were constructed by intercalating the dye between the peptides in the middle of the beta-sheet. The long axis of the dye molecule (in the case of unimolecular systems) or the long axis of the ribbon-like micelle (in the case of the supramolecular dye form) was oriented parallel to the peptide backbone. This positioning maximally reduced the exposure of the hydrophobic diphenyl (central dye fragment) to water. In general the complexes of supramolecular Congo red ligands appeared more stable than those formed by individual dye molecules. Specific interactions (electrostatic and/or ring stacking) dominated as binding forces in the case of the single molecule, while non-specific surface adsorption seemed decisive in complexing with the supramolecular ligand. Both the unimolecular and supramolecular versions of the dye ligand were found to be likely to form complexes of sufficient stability with peptides. The low stability of the protein and the gap accessible to penetration in the peptide sheet seem sufficient for supramolecular ligand binding, but the presence of positively charged or hydrophobic amino acids may strengthen binding significantly. The need for specific interaction makes single-molecule Congo red binding rather unusual as a general amyloid protein ligand. The structural feature of Congo red, which enables specific and common interaction with amyloid proteins, probably derives from the ribbon

Solid-binding peptides: smart tools for nanobiotechnology.

PubMed

Care, Andrew; Bergquist, Peter L; Sunna, Anwar

2015-05-01

Over the past decade, solid-binding peptides (SBPs) have been used increasingly as molecular building blocks in nanobiotechnology. These peptides show selectivity and bind with high affinity to the surfaces of a diverse range of solid materials including metals, metal oxides, metal compounds, magnetic materials, semiconductors, carbon materials, polymers, and minerals. They can direct the assembly and functionalisation of materials, and have the ability to mediate the synthesis and construction of nanoparticles and complex nanostructures. As the availability of newly synthesised nanomaterials expands rapidly, so too do the potential applications for SBPs. Copyright © 2015 Elsevier Ltd. All rights reserved.

NetMHCpan-4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data.

PubMed

Jurtz, Vanessa; Paul, Sinu; Andreatta, Massimo; Marcatili, Paolo; Peters, Bjoern; Nielsen, Morten

2017-11-01

Cytotoxic T cells are of central importance in the immune system's response to disease. They recognize defective cells by binding to peptides presented on the cell surface by MHC class I molecules. Peptide binding to MHC molecules is the single most selective step in the Ag-presentation pathway. Therefore, in the quest for T cell epitopes, the prediction of peptide binding to MHC molecules has attracted widespread attention. In the past, predictors of peptide-MHC interactions have primarily been trained on binding affinity data. Recently, an increasing number of MHC-presented peptides identified by mass spectrometry have been reported containing information about peptide-processing steps in the presentation pathway and the length distribution of naturally presented peptides. In this article, we present NetMHCpan-4.0, a method trained on binding affinity and eluted ligand data leveraging the information from both data types. Large-scale benchmarking of the method demonstrates an increase in predictive performance compared with state-of-the-art methods when it comes to identification of naturally processed ligands, cancer neoantigens, and T cell epitopes. Copyright © 2017 by The American Association of Immunologists, Inc.

Binding of leachable components of polymethyl methacrylate (PMMA) and peptide on modified SPR chip

NASA Astrophysics Data System (ADS)

Szaloki, M.; Vitalyos, G.; Harfalvi, J.; Hegedus, Cs

2013-12-01

Many types of polymers are often used in dentistry, which may cause allergic reaction, mainly methyl methacrylate allergy due to the leachable, degradable components of polymerized dental products. The aim of this study was to investigate the interaction between the leachable components of PMMA and peptides by Fourier-transform Surface Plasmon Resonance (FT SPR). In our previous work binding of oligopeptides (Ph.D.-7 and Ph.D.-12 Peptide Library Kit) was investigated to PMMA surface by phage display technique. It was found that oligopeptides bounded specifically to PMMA surface. The most common amino acids were leucine and proline inside the amino acids sequences of DNA of phages. The binding of haptens, as formaldehyde and methacrylic acid, to frequent amino acids was to investigate on the modified gold SPR chip. Self assembled monolayer (SAM) modified the surface of gold chip and ensured the specific binding between the haptens and amino acids. It was found that amino acids bounded to modified SPR gold and the haptens bounded to amino acids by creating multilayer on the chip surface. By the application of phage display and SPR modern bioanalytical methods the interaction between allergens and peptides can be investigated.

Use of synthetic peptide libraries for the H-2Kd binding motif identification.

PubMed

Quesnel, A; Casrouge, A; Kourilsky, P; Abastado, J P; Trudelle, Y

1995-01-01

To identify Kd-binding peptides, an approach based on small peptide libraries has been developed. These peptide libraries correspond to all possible single-amino acid variants of a particular Kd-binding peptide, SYIPSAEYI, an analog of the Plasmodium berghei 252-260 antigenic peptide SYIPSAEKI. In the parent sequence, each position is replaced by all the genetically encoded amino acids (except cysteine). The multiple analog syntheses are performed either by the Divide Couple and Recombine method or by the Single Resin method and generate mixtures containing 19 peptides. The present report deals with the synthesis, the purification, the chemical characterization by amino acid analysis and electrospray mass spectrometry (ES-MS), and the application of such mixtures in binding tests with a soluble, functionally empty, single-chain H-2Kd molecule denoted SC-Kd. For each mixture, bound peptides were eluted and analyzed by sequencing. Since the binding tests were realized in noncompetitive conditions, our results show that a much broader set of peptides bind to Kd than expected from previous studies. This may be of practical importance when looking for low affinity peptides such as tumor peptides capable of eliciting protective immune response.

A novel Chk1-binding peptide that enhances genotoxic sensitivity through the cellular redistribution of nuclear Chk1

PubMed Central

Kim, Kwang Seok; Choi, Kyu Jin; Bae, Sangwoo

2016-01-01

Since checkpoint kinase 1 (Chk1) is an essential factor for cell viability following DNA damage, the inhibition of Chk1 has been a major focus of pharmaceutical development to enhance the sensitivity of tumor cells to chemo- and radiotherapy that damage DNA. However, due to the off-target effects of conventional Chk1-targeting strategies and the toxicity of Chk1 inhibitors, alternative strategies are required to target Chk1. To facilitate such efforts, in this study, we identified a specific Chk1-binding 12-mer peptide from the screening of a phage display library and characterized the peptide in terms of cellular cytotoxicity, and in terms of its effect on Chk1 activity and sensitivity to genotoxic agents. This peptide, named N-terminal Chk1-binding peptide (Chk1-NP), bound the kinase domain of Chk1. Simulation of the binding revealed that the very N-terminus of the Chk1 kinase domain is the potential peptide binding site. Of note, the polyarginine-mediated internalization of Chk1-NP redistributed nuclear Chk1 with a prominent decrease in the nucleus in the absence of DNA damage. Treatment with Chk1-NP peptide alone decreased the viability of p53-defective HeLa cells, but not that of p53-functional NCI-H460 cells under normal conditions. The treatment of HeLa or NCI-H460 cells with the peptide significantly enhanced radiation sensitivity following ionizing radiation (IR) with a greater enhancement observed in HeLa cells. Moreover, the IR-induced destabilization of Chk1 was aggravated by treatment with Chk1-NP. Therefore, the decreased nuclear localization and protein levels of Chk1 seem to be responsible for the enhanced cancer cell killing following combined treatment with IR and Chk1-NP. The approach using the specific Chk1-binding peptide may facilitate the mechanistic understanding and potential modulation of Chk1 activities and may provide a novel rationale for the development of specific Chk1-targeting agents. PMID:28025997

Binding affinity of pro-apoptotic BH3 peptides for the anti-apoptotic Mcl-1 and A1 proteins: Molecular dynamics simulations of Mcl-1 and A1 in complex with six different BH3 peptides.

PubMed

Modi, Vivek; Sankararamakrishnan, Ramasubbu

2017-05-01

The anti-apoptotic members of Bcl-2 family of proteins bind to their pro-apoptotic counterparts to induce or prevent cell death.Based on the distinct binding profiles for specific pro-apoptotic BH3 peptides, the anti-apoptotic Bcl-2 proteins can be divided into at least two subclasses. The subclass that includes Bcl-X L binds strongly to Bad BH3 peptide while it has weak binding affinity for the second subclass of Bcl-2 proteins such as Mcl-1 and A1. Anti-apoptotic Bcl-2 proteins are considered to be attractive drug targets for anti-cancer drugs. BH3-mimetic inhibitors such as ABT-737 have been shown to be specific to Bcl-X L subclass while Mcl-1 and A1 show resistance to the same drug. An efficacious inhibitor should target all the anti-apoptotic Bcl-2 proteins. Hence, development of inhibitors selective to Mcl-1 and A1 is of prime importance for targeted cancer therapeutics. The first step to achieve this goal is to understand the molecular basis of high binding affinities of specific pro-apoptotic BH3 peptides for Mcl-1 and A1. To understand the interactions between the BH3 peptides and Mcl-1/A1, we performed multi-nanosecond molecular dynamics (MD) simulations of six complex structures of Mcl-1 and A1. With the exception of Bad, all complex structures were experimentally determined. Bad complex structures were modeled. Our simulation studies identified specific pattern of polar interactions between Mcl-1/A1 and high-affinity binding BH3 peptides. The lack of such polar interactions in Bad peptide complex is attributed to specific basic residues present before and after the highly conserved Leu residue. The close approach of basic residues in Bad and Mcl-1/A1 is hypothesized to be the cause of weak binding affinity. To test this hypothesis, we generated in silico mutants of these basic residues in Bad peptide and Mcl-1/A1 proteins. MD simulations of the mutant systems established the pattern of stable polar interactions observed in high-affinity binding BH3

Functional and Selective Bacterial Interfaces Using Cross-Scaffold Gold Binding Peptides

NASA Astrophysics Data System (ADS)

Adams, Bryn L.; Hurley, Margaret M.; Jahnke, Justin P.; Stratis-Cullum, Dimitra N.

2015-11-01

We investigated the functional and selective activity of three phage-derived gold-binding peptides on the Escherichia coli ( E. coli) bacterial cell surface display scaffold (eCPX) for the first time. Gold-binding peptides, p3-Au12 (LKAHLPPSRLPS), p8#9 (VSGSSPDS), and Midas-2 (TGTSVLIATPYV), were compared side-by-side through experiment and simulation. All exhibited strong binding to an evaporated gold film, with approximately a 4-log difference in binding between each peptide and the control sample. The increased affinity for gold was also confirmed by direct visualization of samples using Scanning Electron Microscopy (SEM). Peptide dynamics in solution were performed to analyze innate structure, and all three were found to have a high degree of flexibility. Preferential binding to gold over silicon for all three peptides was demonstrated, with up to four orders of magnitude selectivity exhibited by p3-Au12. The selectivity was also clearly evident through SEM analysis of the boundary between the gold film and silicon substrate. Functional activity of bound E. coli cells was further demonstrated by stimulating filamentation and all three peptides were characterized as prolific relative to control samples. This work shows great promise towards functional and active bacterial-hybrid gold surfaces and the potential to enable the next generation living material interfaces.

Stapled peptide inhibitors of RAB25 target context-specific phenotypes in cancer | Office of Cancer Genomics

Cancer.gov

Recent evidence has established a role for the small GTPase RAB25, as well as related effector proteins, in enacting both pro-oncogenic and anti-oncogenic phenotypes in specific cellular contexts. Here we report the development of all-hydrocarbon stabilized peptides derived from the RAB-binding FIP-family of proteins to target RAB25. Relative to unmodified peptides, optimized stapled peptides exhibit increased structural stability, binding affinity, cell permeability, and inhibition of RAB25:FIP complex formation.

Morintides: cargo-free chitin-binding peptides from Moringa oleifera.

PubMed

Kini, Shruthi G; Wong, Ka H; Tan, Wei Liang; Xiao, Tianshu; Tam, James P

2017-03-31

Hevein-like peptides are a family of cysteine-rich and chitin-binding peptides consisting of 29-45 amino acids. Their chitin-binding property is essential for plant defense against fungi. Based on the number of cysteine residues in their sequences, they are divided into three sub-families: 6C-, 8C- and 10C-hevein-like peptides. All three subfamilies contain a three-domain precursor comprising a signal peptide, a mature hevein-like peptide and a C-terminal domain comprising a hinge region with protein cargo in 8C- and 10C-hevein-like peptides. Here we report the isolation and characterization of two novel 8C-hevein-like peptides, designated morintides (mO1 and mO2), from the drumstick tree Moringa oleifera, a drought-resistant tree belonging to the Moringaceae family. Proteomic analysis revealed that morintides comprise 44 amino acid residues and are rich in cysteine, glycine and hydrophilic amino acid residues such as asparagine and glutamine. Morintides are resistant to thermal and enzymatic degradation, able to bind to chitin and inhibit the growth of phyto-pathogenic fungi. Transcriptomic analysis showed that they contain a three-domain precursor comprising an endoplasmic reticulum (ER) signal sequence, a mature peptide domain and a C-terminal domain. A striking feature distinguishing morintides from other 8C-hevein-like peptides is a short and protein-cargo-free C-terminal domain. Previously, a similar protein-cargo-free C-terminal domain has been observed only in ginkgotides, the 8C-hevein-like peptides from a gymnosperm Ginkgo biloba. Thus, morintides, with a cargo-free C-terminal domain, are a stand-alone class of 8C-hevein-like peptides from angiosperms. Our results expand the existing library of hevein-like peptides and shed light on molecular diversity within the hevein-like peptide family. Our work also sheds light on the anti-fungal activity and stability of 8C-hevein-like peptides.

Enhanced Cellular Adhesion on Titanium by Silk Functionalized with titanium binding and RGD peptides

PubMed Central

Vidal, Guillaume; Blanchi, Thomas; Mieszawska, Aneta J.; Calabrese, Rossella; Rossi, Claire; Vigneron, Pascale; Duval, Jean-Luc; Kaplan, David L.; Egles, Christophe

2012-01-01

Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. Quartz Crystal Microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived RGD peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by Scanning Electron Microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk-peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell-biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required. PMID:22975628

Peptide p5 binds both heparinase-sensitive glycosaminoglycans and fibrils in patient-derived AL amyloid extracts.

PubMed

Martin, Emily B; Williams, Angela; Heidel, Eric; Macy, Sallie; Kennel, Stephen J; Wall, Jonathan S

2013-06-21

In previously published work, we have described heparin-binding synthetic peptides that preferentially recognize amyloid deposits in a mouse model of reactive systemic (AA) amyloidosis and can be imaged by using positron and single photon emission tomographic imaging. We wanted to extend these findings to the most common form of visceral amyloidosis, namely light chain (AL); however, there are no robust experimental animal models of AL amyloidosis. To further define the binding of the lead peptide, p5, to AL amyloid, we characterized the reactivity in vitro of p5 with in situ and patient-derived AL amyloid extracts which contain both hypersulfated heparan sulfate proteoglycans as well as amyloid fibrils. Histochemical staining demonstrated that the peptide specifically localized with tissue-associated AL amyloid deposits. Although we anticipated that p5 would undergo electrostatic interactions with the amyloid-associated glycosaminoglycans expressing heparin-like side chains, no significant correlation between peptide binding and glycosaminoglycan content within amyloid extracts was observed. In contrast, following heparinase I treatment, although overall binding was reduced, a positive correlation between peptide binding and amyloid fibril content became evident. This interaction was further confirmed using synthetic light chain fibrils that contain no carbohydrates. These data suggest that p5 can bind to both the sulfated glycosaminoglycans and protein fibril components of AL amyloid. Understanding these complex electrostatic interactions will aid in the optimization of synthetic peptides for use as amyloid imaging agents and potentially as therapeutics for the treatment of amyloid diseases. Copyright © 2013 Elsevier Inc. All rights reserved.

[Ala12]MCD peptide: a lead peptide to inhibitors of immunoglobulin E binding to mast cell receptors.

PubMed

Buku, A; Condie, B A; Price, J A; Mezei, M

2005-09-01

An effort was made to discover mast cell degranulating (MCD) peptide analogs that bind with high affinity to mast cell receptors without triggering secretion of histamine or other mediators of the allergic reaction initiated by immunoglobulin E (IgE) after mast cell activation. Such compounds could serve as inhibitors of IgE binding to mast cell receptors. An alanine scan of MCD peptide reported previously showed that the analog [Ala12]MCD was 120-fold less potent in histamine-releasing activity and fivefold more potent in binding affinity to mast cell receptors than the parent MCD peptide. Because this analog showed marginal intrinsic activity and good binding affinity it was subsequently tested in the present study as an IgE inhibitor. In contrast to MCD peptide, [Ala12]MCD showed a 50% inhibition of IgE binding to the Fc epsilon RI alpha mast cell receptor by using rat basophilic leukemia (RBL-2H3) mast cells and fluorescence polarization. Furthermore, in a beta-hexosaminidase secretory assay, the peptide also showed a 50% inhibition of the secretion of this enzyme caused by IgE. An attempt was made to relate structural changes and biologic differences between the [Ala12]MCD analog and the parent MCD peptide. The present results show that [Ala12]MCD may provide a base for designing agents to prevent IgE/Fc epsilon RI alpha interactions and, consequently, allergic conditions.

Dual-functioning peptides discovered by phage display increase the magnitude and specificity of BMSC attachment to mineralized biomaterials.

PubMed

Ramaraju, Harsha; Miller, Sharon J; Kohn, David H

2017-07-01

Design of biomaterials for cell-based therapies requires presentation of specific physical and chemical cues to cells, analogous to cues provided by native extracellular matrices (ECM). We previously identified a peptide sequence with high affinity towards apatite (VTKHLNQISQSY, VTK) using phage display. The aims of this study were to identify a human MSC-specific peptide sequence through phage display, combine it with the apatite-specific sequence, and verify the specificity of the combined dual-functioning peptide to both apatite and human bone marrow stromal cells. In this study, a combinatorial phage display identified the cell binding sequence (DPIYALSWSGMA, DPI) which was combined with the mineral binding sequence to generate the dual peptide DPI-VTK. DPI-VTK demonstrated significantly greater binding affinity (1/K D ) to apatite surfaces compared to VTK, phosphorylated VTK (VTK phos ), DPI-VTK phos , RGD-VTK, and peptide-free apatite surfaces (p < 0.01), while significantly increasing hBMSC adhesion strength (τ 50 , p < 0.01). MSCs demonstrated significantly greater adhesion strength to DPI-VTK compared to other cell types, while attachment of MC3T3 pre-osteoblasts and murine fibroblasts was limited (p < 0.01). MSCs on DPI-VTK coated surfaces also demonstrated increased spreading compared to pre-osteoblasts and fibroblasts. MSCs cultured on DPI-VTK coated apatite films exhibited significantly greater proliferation compared to controls (p < 0.001). Moreover, early and late stage osteogenic differentiation markers were elevated on DPI-VTK coated apatite films compared to controls. Taken together, phage display can identify non-obvious cell and material specific peptides to increase human MSC adhesion strength to specific biomaterial surfaces and subsequently increase cell proliferation and differentiation. These new peptides expand biomaterial design methodology for cell-based regeneration of bone defects. This strategy of combining cell and material

Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

PubMed

Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

2015-09-01

Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Structural and thermodynamic characterization of the recognition of the S100-binding peptides TRTK12 and p53 by calmodulin

PubMed Central

Wafer, Lucas N; Tzul, Franco O; Pandharipande, Pranav P; McCallum, Scott A; Makhatadze, George I

2014-01-01

Calmodulin (CaM) is a multifunctional messenger protein that activates a wide variety of signaling pathways in eukaryotic cells in a calcium-dependent manner. CaM has been proposed to be functionally distinct from the S100 proteins, a related family of eukaryotic calcium-binding proteins. Previously, it was demonstrated that peptides derived from the actin-capping protein, TRTK12, and the tumor-suppressor protein, p53, interact with multiple members of the S100 proteins. To test the specificity of these peptides, they were screened using isothermal titration calorimetry against 16 members of the human S100 protein family, as well as CaM, which served as a negative control. Interestingly, both the TRTK12 and p53 peptides were found to interact with CaM. These interactions were further confirmed by both fluorescence and nuclear magnetic resonance spectroscopies. These peptides have distinct sequences from the known CaM target sequences. The TRTK12 peptide was found to independently interact with both CaM domains and bind with a stoichiometry of 2:1 and dissociations constants Kd,C-term = 2 ± 1 µM and Kd,N-term = 14 ± 1 µM. In contrast, the p53 peptide was found to interact only with the C-terminal domain of CaM, Kd,C-term =2 ± 1 µM, 25°C. Using NMR spectroscopy, the locations of the peptide binding sites were mapped onto the structure of CaM. The binding sites for both peptides were found to overlap with the binding interface for previously identified targets on both domains of CaM. This study demonstrates the plasticity of CaM in target binding and may suggest a possible overlap in target specificity between CaM and the S100 proteins. PMID:24947426

Protonation States in molecular dynamics simulations of peptide folding and binding.

PubMed

Ben-Shimon, Avraham; Shalev, Deborah E; Niv, Masha Y

2013-01-01

Peptides are important signaling modules, acting both as individual hormones and as parts of larger molecules, mediating their protein-protein interactions. Many peptidic and peptidomimetic drugs have reached the marketplace and opportunities for peptide-based drug discovery are on the rise. pH-dependent behavior of peptides is well documented in the context of misfolding diseases and peptide translocation. Changes in the protonation states of peptide residues often have a crucial effect on a peptide's structure, dynamics and function, which may be exploited for biotechnological applications. The current review surveys the increasing levels of sophistication in the treatment of protonation states in computational studies involving peptides. Specifically we describe I) the common practice of assigning a single protonation state and using it throughout the dynamic simulation, II) approaches that consider multiple protonation states and compare computed observables to experimental ones, III) constant pH molecular dynamics methods that couple changes in protonation states with conformational dynamics "on the fly". Applications of conformational dynamics treatment of peptides in the context of binding, folding and interactions with the membrane are presented, illustrating the growing body of work in this field and highlighting the importance of careful handling of protonation states of peptidic residues.

Demonstration of a Specific Escherichia coli SecY–Signal Peptide Interaction†

PubMed Central

Wang, Ligong; Miller, Alexander; Rusch, Sharyn L.; Kendall, Debra A.

2011-01-01

Protein translocation in Escherichia coli is initiated by the interaction of a preprotein with the membrane translocase composed of a motor protein, SecA ATPase, and a membrane-embedded channel, the SecYEG complex. The extent to which the signal peptide region of the preprotein plays a role in SecYEG interactions is unclear, in part because studies in this area typically employ the entire preprotein. Using a synthetic signal peptide harboring a photoaffinity label in its hydrophobic core, we examined this interaction with SecYEG in a detergent micellar environment. The signal peptide was found to specifically bind SecY in a saturable manner and at levels comparable to those that stimulate SecA ATPase activity. Chemical and proteolytic cleavage of cross-linked SecY and analysis of the signal peptide adducts indicate that the binding was primarily to regions of the protein containing transmembrane domains seven and two. The signal peptide–SecY interaction was affected by the presence of SecA and nucleotides in a manner consistent with the transfer of signal peptide to SecY upon nucleotide hydrolysis at SecA. PMID:15476412

Peptide-binding motifs of two common equine class I MHC molecules in Thoroughbred horses.

PubMed

Bergmann, Tobias; Lindvall, Mikaela; Moore, Erin; Moore, Eugene; Sidney, John; Miller, Donald; Tallmadge, Rebecca L; Myers, Paisley T; Malaker, Stacy A; Shabanowitz, Jeffrey; Osterrieder, Nikolaus; Peters, Bjoern; Hunt, Donald F; Antczak, Douglas F; Sette, Alessandro

2017-05-01

Quantitative peptide-binding motifs of MHC class I alleles provide a valuable tool to efficiently identify putative T cell epitopes. Detailed information on equine MHC class I alleles is still very limited, and to date, only a single equine MHC class I allele, Eqca-1*00101 (ELA-A3 haplotype), has been characterized. The present study extends the number of characterized ELA class I specificities in two additional haplotypes found commonly in the Thoroughbred breed. Accordingly, we here report quantitative binding motifs for the ELA-A2 allele Eqca-16*00101 and the ELA-A9 allele Eqca-1*00201. Utilizing analyses of endogenously bound and eluted ligands and the screening of positional scanning combinatorial libraries, detailed and quantitative peptide-binding motifs were derived for both alleles. Eqca-16*00101 preferentially binds peptides with aliphatic/hydrophobic residues in position 2 and at the C-terminus, and Eqca-1*00201 has a preference for peptides with arginine in position 2 and hydrophobic/aliphatic residues at the C-terminus. Interestingly, the Eqca-16*00101 motif resembles that of the human HLA A02-supertype, while the Eqca-1*00201 motif resembles that of the HLA B27-supertype and two macaque class I alleles. It is expected that the identified motifs will facilitate the selection of candidate epitopes for the study of immune responses in horses.

Identification of tissue-specific targeting peptide

NASA Astrophysics Data System (ADS)

Jung, Eunkyoung; Lee, Nam Kyung; Kang, Sang-Kee; Choi, Seung-Hoon; Kim, Daejin; Park, Kisoo; Choi, Kihang; Choi, Yun-Jaie; Jung, Dong Hyun

2012-11-01

Using phage display technique, we identified tissue-targeting peptide sets that recognize specific tissues (bone-marrow dendritic cell, kidney, liver, lung, spleen and visceral adipose tissue). In order to rapidly evaluate tissue-specific targeting peptides, we performed machine learning studies for predicting the tissue-specific targeting activity of peptides on the basis of peptide sequence information using four machine learning models and isolated the groups of peptides capable of mediating selective targeting to specific tissues. As a representative liver-specific targeting sequence, the peptide "DKNLQLH" was selected by the sequence similarity analysis. This peptide has a high degree of homology with protein ligands which can interact with corresponding membrane counterparts. We anticipate that our models will be applicable to the prediction of tissue-specific targeting peptides which can recognize the endothelial markers of target tissues.

The P9 peptide sidechain specificity of I-Ad.

PubMed

Bartnes, K; Li, X; Briand, J P; Travers, P J; Hannestad, K

1999-12-01

The murine MHC class II variant I-Ad confers susceptibility to herpes simplex virus (HSV)-induced keratitis and relative protection against type 1 diabetes mellitus. The association to these autoimmune diseases appears to be largely determined by the peptide sidechain specificity of the P9 pocket, which we therefore have analyzed in detail. Assessment of T-cell responses and I-Ad binding capacity of position 446-substituted analogs of an IgG2a allotype b (IgG2a(b)) heavy chain peptide demonstrates that engagement of the P9 pocket is crucial for effective peptide presentation. Sidechain size rather than charge decides the capacity to engage the P9 pocket. Thus, small, uncharged sidechains are accepted, whereas acidic and aromatic amino acids as well as lysine and arginine are disfavored. The specificity of the P9 pocket of I-Ad (serine beta57) is distinct from that of the diabetes-associated I-Ag7 (aspartic acid beta57), supporting the contention that the polymorphism at residue beta57 influences diabetes susceptibility via P9-specific effects on the repertoires of self peptides presented to T cells. Furthermore, the data rationalize the susceptibility to HSV-induced keratitis conferred by the a and the protection conferred by the b allotypes of the IgG2a heavy chain. Keratitogenic T cells, which cross-react with the viral UL6 protein and a corneal antigen, are silenced in IgG2a(b) mice because of antigenic mimicry with gamma2a(b) 435-451. Our finding that the lysine P9 residue of the corresponding gamma2a(a) allopeptide precludes high-affinity binding to I-Ad indicates that the susceptibility of IgG2a(a) mice reflects inefficient thymic presentation of autologous IgG2a and thus failure to purge the T-cell repertoire of the pathogenic clones.

Structure of the mouse sex peptide pheromone ESP1 reveals a molecular basis for specific binding to the class C G-protein-coupled vomeronasal receptor.

PubMed

Yoshinaga, Sosuke; Sato, Toru; Hirakane, Makoto; Esaki, Kaori; Hamaguchi, Takashi; Haga-Yamanaka, Sachiko; Tsunoda, Mai; Kimoto, Hiroko; Shimada, Ichio; Touhara, Kazushige; Terasawa, Hiroaki

2013-05-31

Exocrine gland-secreting peptide 1 (ESP1) is a sex pheromone that is released in male mouse tear fluids and enhances female sexual receptive behavior. ESP1 is selectively recognized by a specific class C G-protein-coupled receptor (GPCR), V2Rp5, among the hundreds of receptors expressed in vomeronasal sensory neurons (VSNs). The specific sensing mechanism of the mammalian peptide pheromone by the class C GPCR remains to be elucidated. Here we identified the minimal functional region needed to retain VSN-stimulating activity in ESP1 and determined its three-dimensional structure, which adopts a helical fold stabilized by an intramolecular disulfide bridge with extensive charged patches. We then identified the amino acids involved in the activation of VSNs by a structure-based mutational analysis, revealing that the highly charged surface is crucial for the ESP1 activity. We also demonstrated that ESP1 specifically bound to an extracellular region of V2Rp5 by an in vitro pulldown assay. Based on homology modeling of V2Rp5 using the structure of the metabotropic glutamate receptor, we constructed a docking model of the ESP1-V2Rp5 complex in which the binding interface exhibited good electrostatic complementarity. These experimental results, supported by the molecular docking simulations, reveal that charge-charge interactions determine the specificity of ESP1 binding to V2Rp5 in the large extracellular region characteristic of class C GPCRs. The present study provides insights into the structural basis for the narrowly tuned sensing of mammalian peptide pheromones by class C GPCRs.

Automated benchmarking of peptide-MHC class I binding predictions.

PubMed

Trolle, Thomas; Metushi, Imir G; Greenbaum, Jason A; Kim, Yohan; Sidney, John; Lund, Ole; Sette, Alessandro; Peters, Bjoern; Nielsen, Morten

2015-07-01

Numerous in silico methods predicting peptide binding to major histocompatibility complex (MHC) class I molecules have been developed over the last decades. However, the multitude of available prediction tools makes it non-trivial for the end-user to select which tool to use for a given task. To provide a solid basis on which to compare different prediction tools, we here describe a framework for the automated benchmarking of peptide-MHC class I binding prediction tools. The framework runs weekly benchmarks on data that are newly entered into the Immune Epitope Database (IEDB), giving the public access to frequent, up-to-date performance evaluations of all participating tools. To overcome potential selection bias in the data included in the IEDB, a strategy was implemented that suggests a set of peptides for which different prediction methods give divergent predictions as to their binding capability. Upon experimental binding validation, these peptides entered the benchmark study. The benchmark has run for 15 weeks and includes evaluation of 44 datasets covering 17 MHC alleles and more than 4000 peptide-MHC binding measurements. Inspection of the results allows the end-user to make educated selections between participating tools. Of the four participating servers, NetMHCpan performed the best, followed by ANN, SMM and finally ARB. Up-to-date performance evaluations of each server can be found online at http://tools.iedb.org/auto_bench/mhci/weekly. All prediction tool developers are invited to participate in the benchmark. Sign-up instructions are available at http://tools.iedb.org/auto_bench/mhci/join. mniel@cbs.dtu.dk or bpeters@liai.org Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Automated benchmarking of peptide-MHC class I binding predictions

PubMed Central

Trolle, Thomas; Metushi, Imir G.; Greenbaum, Jason A.; Kim, Yohan; Sidney, John; Lund, Ole; Sette, Alessandro; Peters, Bjoern; Nielsen, Morten

2015-01-01

Motivation: Numerous in silico methods predicting peptide binding to major histocompatibility complex (MHC) class I molecules have been developed over the last decades. However, the multitude of available prediction tools makes it non-trivial for the end-user to select which tool to use for a given task. To provide a solid basis on which to compare different prediction tools, we here describe a framework for the automated benchmarking of peptide-MHC class I binding prediction tools. The framework runs weekly benchmarks on data that are newly entered into the Immune Epitope Database (IEDB), giving the public access to frequent, up-to-date performance evaluations of all participating tools. To overcome potential selection bias in the data included in the IEDB, a strategy was implemented that suggests a set of peptides for which different prediction methods give divergent predictions as to their binding capability. Upon experimental binding validation, these peptides entered the benchmark study. Results: The benchmark has run for 15 weeks and includes evaluation of 44 datasets covering 17 MHC alleles and more than 4000 peptide-MHC binding measurements. Inspection of the results allows the end-user to make educated selections between participating tools. Of the four participating servers, NetMHCpan performed the best, followed by ANN, SMM and finally ARB. Availability and implementation: Up-to-date performance evaluations of each server can be found online at http://tools.iedb.org/auto_bench/mhci/weekly. All prediction tool developers are invited to participate in the benchmark. Sign-up instructions are available at http://tools.iedb.org/auto_bench/mhci/join. Contact: mniel@cbs.dtu.dk or bpeters@liai.org Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25717196

A nonpolymorphic major histocompatibility complex class Ib molecule binds a large array of diverse self-peptides

PubMed Central

1994-01-01

Unlike the highly polymorphic major histocompatibility complex (MHC) class Ia molecules, which present a wide variety of peptides to T cells, it is generally assumed that the nonpolymorphic MHC class Ib molecules may have evolved to function as highly specialized receptors for the presentation of structurally unique peptides. However, a thorough biochemical analysis of one class Ib molecule, the soluble isoform of Qa-2 antigen (H-2SQ7b), has revealed that it binds a diverse array of structurally similar peptides derived from intracellular proteins in much the same manner as the classical antigen-presenting molecules. Specifically, we find that SQ7b molecules are heterodimers of heavy and light chains complexed with nonameric peptides in a 1:1:1 ratio. These peptides contain a conserved hydrophobic residue at the COOH terminus and a combination of one or more conserved residue(s) at P7 (histidine), P2 (glutamine/leucine), and/or P3 (leucine/asparagine) as anchors for binding SQ7b. 2 of 18 sequenced peptides matched cytosolic proteins (cofilin and L19 ribosomal protein), suggesting an intracellular source of the SQ7b ligands. Minimal estimates of the peptide repertoire revealed that at least 200 different naturally processed self-peptides can bind SQ7b molecules. Since Qa-2 molecules associate with a diverse array of peptides, we suggest that they function as effective presenting molecules of endogenously synthesized proteins like the class Ia molecules. PMID:8294869

Use of a Phage-Display Method to Identify Peptides that Bind to a Tin Oxide Nanosheets.

PubMed

Nakazawa, Hikaru; Seta, Yasuko; Hirose, Tatsuya; Masuda, Yoshitake; Umetsu, Mitsuo

2018-01-01

Nanosheets of SnO2 which an n-type semiconductor with a rutile-type crystalline structure are predominantly used as gas sensors. SnO2 nanosheets have a tetragonal crystal structure where growth along the c-axis is suppressed to form a sheet. The major exposed facets of SnO2 nanosheets have {110}, {101} and {211} crystal planes along the a-axis, with the reduced {110} surface having a particularly high surface energy. Identifying peptides that bind to specific crystal planes by using peptide phage-display approach will increase the potential applications of metal oxide nanomaterials by fusing proteins with desirable active sites to peptides that adsorb at high density on the major exposed crystal plane of nanosheets. It may be possible to construct highly sensitive biosensors. The main objective of the present study is to identify peptides that adsorb preferentially to a SnO2 nanosheet by using peptide-phage display approach. Four milligrams of SnO2 nanosheet were mixed with 1011 plaque-forming units of Ph.D.-12 Phage Display Peptide Library. Phage-bound nanosheet particles were washed 10 times with 1 mL of phosphatebuffered saline containing 0.5% Tween 20. Phages bound to the nanosheet were eluted with three different buffers: (1) high-salt buffer containing 2 M NaCl (pH 7.5); (2) acidic buffer containing 200 mM Gly-HCl (pH 2.2); and (3) high-phosphate-ion buffer containing 500 mM NaH2PO4 (pH 7.5). The eluted phages were subjected to four or five rounds of biopanning. At each round, individual plaques were picked from the plates, and the amino acid sequences of the peptides were identified by DNA sequencing. The identified SnO2-binding peptides labeled with fluorescein isothiocyanate were synthesized. Adsorption isotherms were constructed at peptide concentrations ranging from 0.25 to 2.0 µM with 4mg of nanomaterials. We were determined the sequences of 11 clones with the high-salt buffer, 7 with the high-phosphateion buffers, and 6 with the acidic buffer and

Access to site-specific Fc-cRGD peptide conjugates through streamlined expressed protein ligation.

PubMed

Frutos, S; Jordan, J B; Bio, M M; Muir, T W; Thiel, O R; Vila-Perelló, M

2016-10-12

An ideal drug should be highly effective, non-toxic and be delivered by a convenient and painless single dose. We are still far from such optimal treatment but peptides, with their high target selectivity and low toxicity profiles, provide a very attractive platform from which to strive towards it. One of the major limitations of peptide drugs is their high clearance rates, which limit dosage regimen options. Conjugation to antibody Fc domains is a viable strategy to improve peptide stability by increasing their hydrodynamic radius and hijacking the Fc recycling pathway. We report the use of a split-intein based semi-synthetic approach to site-specifically conjugate a synthetic integrin binding peptide to an Fc domain. The strategy described here allows conjugating synthetic peptides to Fc domains, which is not possible via genetic methods, fully maintaining the ability of both the Fc domain and the bioactive peptide to interact with their binding partners.

Access to site-specific Fc–cRGD peptide conjugates through streamlined expressed protein ligation†

PubMed Central

Frutos, S.; Jordan, J. B.; Bio, M. M.; Muir, T. W.; Thiel, O. R.; Vila-Perelló, M.

2018-01-01

An ideal drug should be highly effective, non-toxic and be delivered by a convenient and painless single dose. We are still far from such optimal treatment but peptides, with their high target selectivity and low toxicity profiles, provide a very attractive platform from which to strive towards it. One of the major limitations of peptide drugs is their high clearance rates, which limit dosage regimen options. Conjugation to antibody Fc domains is a viable strategy to improve peptide stability by increasing their hydrodynamic radius and hijacking the Fc recycling pathway. We report the use of a split-intein based semi-synthetic approach to site-specifically conjugate a synthetic integrin binding peptide to an Fc domain. The strategy described here allows conjugating synthetic peptides to Fc domains, which is not possible via genetic methods, fully maintaining the ability of both the Fc domain and the bioactive peptide to interact with their binding partners. PMID:27722696

Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.

PubMed

Ma, Xianyue; Cline, Kenneth

2013-03-01

Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC-cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.

Use of superparamagnetic beads for the isolation of a peptide with specificity to cymbidium mosaic virus.

PubMed

Ooi, Diana Jia Miin; Dzulkurnain, Adriya; Othman, Rofina Yasmin; Lim, Saw Hoon; Harikrishna, Jennifer Ann

2006-09-01

A modified method for the rapid isolation of specific ligands to whole virus particles is described. Biopanning against cymbidium mosaic virus was carried out with a commercial 12-mer random peptide display library. A solution phase panning method was devised using streptavidin-coated superparamagnetic beads. The solution based panning method was more efficient than conventional immobilized target panning when using whole viral particles of cymbidium mosaic virus as a target. Enzyme-linked immunosorbent assay of cymbidium mosaic virus-binding peptides isolated from the library identified seven peptides with affinity for cymbidium mosaic virus and one peptide which was specific to cymbidium mosaic virus and had no significant binding to odontoglossum ringspot virus. This method should have broad application for the screening of whole viral particles towards the rapid development of diagnostic reagents without the requirement for cloning and expression of single antigens.

In vivo imaging of hepatocellular carcinoma using a glypican-3-binding peptide based probe

NASA Astrophysics Data System (ADS)

Zhang, Qi; Han, Zhihao; Zhang, Wancun; Qian, Zhiyu; Gu, Yueqing

2017-02-01

Hepatocellular carcinoma (HCC) has been the third most common cause of cancer-related death worldwide. Glypican-3 (GPC3) is a heparin sulfate proteoglycan linked to the cell membrane by a glycosyl-phosphatidylinositol anchor (GPI) and is expressed by 75% of all hepatocellular carcinomas but undetectable in healthy liver tissue or liver with focal lesions. What's more, GPC3 has been gradually applied in clinical applications as a specific indicator for the early detection and prognosis of HCC. As GPC3 can also regulate many pathways in HCC pathogenesis including Wnt, Hh and Yap signaling, it has been shown that GPC3 knockdown can inhibit HCC growth, reinforcing the important roles of GPC3 in HCC development. For HCC early detection, we designed a peptide targeting GPC3 that allows to establish a fluorescent dyes-labeled probe. Firstly, according to the structure of the GPC3 antibody GC33 and the positive peptide reported in the literature, we generated a peptide consisting of twelve amino acids named 12P that may bind to GPC3 with tight binding ability and specificity. In vitro testing, we utilized FCM and laser confocal microscopy to verify its specificity of targeting to the high expression cells of GPC3. What's more, we linked 12P with a near infrared dye to verify its in vivo targeting ability. All results indicated that 12P possessed potent binding capacity which could be used as a targeting module in GPC3 detection probe.

Protein nanopore-based, single-molecule exploration of copper binding to an antimicrobial-derived, histidine-containing chimera peptide.

PubMed

Mereuta, Loredana; Schiopu, Irina; Asandei, Alina; Park, Yoonkyung; Hahm, Kyung-Soo; Luchian, Tudor

2012-12-11

Metal ions binding exert a crucial influence upon the aggregation properties and stability of peptides, and the propensity of folding in various substates. Herein, we demonstrate the use of the α-HL protein as a powerful nanoscopic tool to probe Cu(2+)-triggered physicochemical changes of a 20 aminoacids long, antimicrobial-derived chimera peptide with a His residue as metal-binding site, and simultaneously dissect the kinetics of the free- and Cu(2+)-bound peptide interaction to the α-HL pore. Combining single-molecule electrophysiology on reconstituted lipid membranes and fluorescence spectroscopy, we show that the association rate constant between the α-HL pore and a Cu(2+)-free peptide is higher than that of a Cu(2+)-complexed peptide. We posit that mainly due to conformational changes induced by the bound Cu(2+) on the peptide, the resulting complex encounters a higher energy barrier toward its association with the protein pore, stemming most likely from an extra entropy cost needed to fit the Cu(2+)-complexed peptide within the α-HL lumen region. The lower dissociation rate constant of the Cu(2+)-complexed peptide from α-HL pore, as compared to that of Cu(2+)-free peptide, supports the existence of a deeper free energy well for the protein interaction with a Cu(2+)-complexed peptide, which may be indicative of specific Cu(2+)-mediated contributions to the binding of the Cu(2+)-complexed peptide within the pore lumen.

sNebula, a network-based algorithm to predict binding between human leukocyte antigens and peptides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luo, Heng; Ye, Hao; Ng, Hui Wen

Understanding the binding between human leukocyte antigens (HLAs) and peptides is important to understand the functioning of the immune system. Since it is time-consuming and costly to measure the binding between large numbers of HLAs and peptides, computational methods including machine learning models and network approaches have been developed to predict HLA-peptide binding. However, there are several limitations for the existing methods. We developed a network-based algorithm called sNebula to address these limitations. We curated qualitative Class I HLA-peptide binding data and demonstrated the prediction performance of sNebula on this dataset using leave-one-out cross-validation and five-fold cross-validations. Furthermore, this algorithmmore » can predict not only peptides of different lengths and different types of HLAs, but also the peptides or HLAs that have no existing binding data. We believe sNebula is an effective method to predict HLA-peptide binding and thus improve our understanding of the immune system.« less

sNebula, a network-based algorithm to predict binding between human leukocyte antigens and peptides

PubMed Central

Luo, Heng; Ye, Hao; Ng, Hui Wen; Sakkiah, Sugunadevi; Mendrick, Donna L.; Hong, Huixiao

2016-01-01

Understanding the binding between human leukocyte antigens (HLAs) and peptides is important to understand the functioning of the immune system. Since it is time-consuming and costly to measure the binding between large numbers of HLAs and peptides, computational methods including machine learning models and network approaches have been developed to predict HLA-peptide binding. However, there are several limitations for the existing methods. We developed a network-based algorithm called sNebula to address these limitations. We curated qualitative Class I HLA-peptide binding data and demonstrated the prediction performance of sNebula on this dataset using leave-one-out cross-validation and five-fold cross-validations. This algorithm can predict not only peptides of different lengths and different types of HLAs, but also the peptides or HLAs that have no existing binding data. We believe sNebula is an effective method to predict HLA-peptide binding and thus improve our understanding of the immune system. PMID:27558848

sNebula, a network-based algorithm to predict binding between human leukocyte antigens and peptides

DOE PAGES

Luo, Heng; Ye, Hao; Ng, Hui Wen; ...

2016-08-25

Understanding the binding between human leukocyte antigens (HLAs) and peptides is important to understand the functioning of the immune system. Since it is time-consuming and costly to measure the binding between large numbers of HLAs and peptides, computational methods including machine learning models and network approaches have been developed to predict HLA-peptide binding. However, there are several limitations for the existing methods. We developed a network-based algorithm called sNebula to address these limitations. We curated qualitative Class I HLA-peptide binding data and demonstrated the prediction performance of sNebula on this dataset using leave-one-out cross-validation and five-fold cross-validations. Furthermore, this algorithmmore » can predict not only peptides of different lengths and different types of HLAs, but also the peptides or HLAs that have no existing binding data. We believe sNebula is an effective method to predict HLA-peptide binding and thus improve our understanding of the immune system.« less

Prediction of the binding affinities of peptides to class II MHC using a regularized thermodynamic model

PubMed Central

2010-01-01

Background The binding of peptide fragments of extracellular peptides to class II MHC is a crucial event in the adaptive immune response. Each MHC allotype generally binds a distinct subset of peptides and the enormous number of possible peptide epitopes prevents their complete experimental characterization. Computational methods can utilize the limited experimental data to predict the binding affinities of peptides to class II MHC. Results We have developed the Regularized Thermodynamic Average, or RTA, method for predicting the affinities of peptides binding to class II MHC. RTA accounts for all possible peptide binding conformations using a thermodynamic average and includes a parameter constraint for regularization to improve accuracy on novel data. RTA was shown to achieve higher accuracy, as measured by AUC, than SMM-align on the same data for all 17 MHC allotypes examined. RTA also gave the highest accuracy on all but three allotypes when compared with results from 9 different prediction methods applied to the same data. In addition, the method correctly predicted the peptide binding register of 17 out of 18 peptide-MHC complexes. Finally, we found that suboptimal peptide binding registers, which are often ignored in other prediction methods, made significant contributions of at least 50% of the total binding energy for approximately 20% of the peptides. Conclusions The RTA method accurately predicts peptide binding affinities to class II MHC and accounts for multiple peptide binding registers while reducing overfitting through regularization. The method has potential applications in vaccine design and in understanding autoimmune disorders. A web server implementing the RTA prediction method is available at http://bordnerlab.org/RTA/. PMID:20089173

Discovery and application of peptides that bind to proteins and solid state inorganic materials

NASA Astrophysics Data System (ADS)

Stearns, Linda A.

A series of three projects was undertaken on the theme of peptide-based molecular recognition. In the first project, a messenger RNA (mRNA) display selection was carried out against the II-VI semiconductors zinc sulfide (ZnS), zinc selenide (ZnSe), and cadmium sulfide (CdS). Sequence analysis of 18-mer semiconductor-binding peptides (SBPs) following four rounds of selection indicated that the amino acid sequences were enriched in polar residues compared to the naive library, suggesting that hydrogen-bonding interactions are a dominant mode of interaction between the SBPs and their cognate inorganic surfaces. Select peptides were expressed as fusions of the green fluorescent protein (GFP) to visualize their recognition of semiconductor crystals. Interpretation of the results was complicated by a high fluorescence background that was observed with certain control GFP fusions. Additional experiments, including cross-specificity binding assays, are needed to characterize the peptides that were isolated in this selection. A second project described the practical application of a known inorganic-binding and nucleating peptide. Peptide A3, which was previously isolated by phage display, was chemically conjugated to a short DNA strand using the heterobifunctional linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC). The resulting peptide-DNA conjugate was hybridized to ten complementary single-stranded capture probes extending outward from the surface of an origami DNA nanotube. A gold precursor solution was added to initiate nucleation and growth of gold nanoparticles at the site of the peptide. Transmission electron microscopy (TEM) was used to visualize the gold nanoparticle-decorated nanostructures. This approach holds immense promise for organizing compositionally-diverse materials at the nanoscale. In a third project, a novel non-iterative approach to mRNA display called covalent capture was demonstrated. Using human transferrin as a target

Penetration of short fluorescence-labeled peptides into the nucleus in HeLa cells and in vitro specific interaction of the peptides with deoxyribooligonucleotides and DNA.

PubMed

Fedoreyeva, L I; Kireev, I I; Khavinson, V Kh; Vanyushin, B F

2011-11-01

Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.

Prediction of Surface and pH-Specific Binding of Peptides to Metal and Oxide Nanoparticles

NASA Astrophysics Data System (ADS)

Heinz, Hendrik; Lin, Tzu-Jen; Emami, Fateme Sadat; Ramezani-Dakhel, Hadi; Naik, Rajesh; Knecht, Marc; Perry, Carole C.; Huang, Yu

2015-03-01

The mechanism of specific peptide adsorption onto metallic and oxidic nanostructures has been elucidated in atomic resolution using novel force fields and surface models in comparison to measurements. As an example, variations in peptide adsorption on Pd and Pt nanoparticles depending on shape, size, and location of peptides on specific bounding facets are explained. Accurate computational predictions of reaction rates in C-C coupling reactions using particle models derived from HE-XRD and PDF data illustrate the utility of computational methods for the rational design of new catalysts. On oxidic nanoparticles such as silica and apatites, it is revealed how changes in pH lead to similarity scores of attracted peptides lower than 20%, supported by appropriate model surfaces and data from adsorption isotherms. The results demonstrate how new computational methods can support the design of nanoparticle carriers for drug release and the understanding of calcification mechanisms in the human body.

Increased Peptide Contacts Govern High Affinity Binding of a Modified TCR Whilst Maintaining a Native pMHC Docking Mode

PubMed Central

Cole, David K.; Sami, Malkit; Scott, Daniel R.; Rizkallah, Pierre J.; Borbulevych, Oleg Y.; Todorov, Penio T.; Moysey, Ruth K.; Jakobsen, Bent K.; Boulter, Jonathan M.; Baker, Brian M.; Yi Li

2013-01-01

Natural T cell receptors (TCRs) generally bind to their cognate pMHC molecules with weak affinity and fast kinetics, limiting their use as therapeutic agents. Using phage display, we have engineered a high affinity version of the A6 wild-type TCR (A6wt), specific for the human leukocyte antigen (HLA-A∗0201) complexed with human T cell lymphotropic virus type 111–19 peptide (A2-Tax). Mutations in just 4 residues in the CDR3β loop region of the A6wt TCR were selected that improved binding to A2-Tax by nearly 1000-fold. Biophysical measurements of this mutant TCR (A6c134) demonstrated that the enhanced binding was derived through favorable enthalpy and a slower off-rate. The structure of the free A6c134 TCR and the A6c134/A2-Tax complex revealed a native binding mode, similar to the A6wt/A2-Tax complex. However, concordant with the more favorable binding enthalpy, the A6c134 TCR made increased contacts with the Tax peptide compared with the A6wt/A2-Tax complex, demonstrating a peptide-focused mechanism for the enhanced affinity that directly involved the mutated residues in the A6c134 TCR CDR3β loop. This peptide-focused enhanced TCR binding may represent an important approach for developing antigen specific high affinity TCR reagents for use in T cell based therapies. PMID:23805144

Evaluation of Phage Display Discovered Peptides as Ligands for Prostate-Specific Membrane Antigen (PSMA)

PubMed Central

Edwards, W. Barry

2013-01-01

The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities KD∼1 µM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy. PMID:23935860

Antifreeze and cryoprotective activities of ice-binding collagen peptides from pig skin.

PubMed

Cao, Hui; Zhao, Ying; Zhu, Yu Bing; Xu, Fei; Yu, Jing Song; Yuan, Min

2016-03-01

A novel "hyperactive" ice-binding peptide from porcine collagen was prepared by alkaline protease hydrolysis and a series of column chromatography separations, and then its antifreeze and cryoprotective properties were reported. Using differential scanning calorimetry (DSC), the thermal hysteresis (TH) of ice-binding collagen peptides was closely related to their concentration and crystal fraction. Collagen hydrolysates with maximal TH were obtained by hydrolysis at pH 8.0, DH 15.0%, and 5% alkaline protease at 55°C. After purification by column chromatography, the AP-3 ice-binding collagen peptide (GLLGPLGPRGLL) with 1162.8Da molecular weights exhibited the highest TH (5.28°C), which can be classified as "hyperactive". Recrystallisation and melt-resistance of ice cream were improved by AP-3 ice-binding collagen peptide at 0.2% (w/v) in a similar manner to natural antifreeze proteins. Moreover, the addition of AP-3 collagen peptides in ice cream greatly elevated the glass transition temperature (Tg) to -17.64°C. Copyright © 2015 Elsevier Ltd. All rights reserved.

Prediction of Nucleotide Binding Peptides Using Star Graph Topological Indices.

PubMed

Liu, Yong; Munteanu, Cristian R; Fernández Blanco, Enrique; Tan, Zhiliang; Santos Del Riego, Antonino; Pazos, Alejandro

2015-11-01

The nucleotide binding proteins are involved in many important cellular processes, such as transmission of genetic information or energy transfer and storage. Therefore, the screening of new peptides for this biological function is an important research topic. The current study proposes a mixed methodology to obtain the first classification model that is able to predict new nucleotide binding peptides, using only the amino acid sequence. Thus, the methodology uses a Star graph molecular descriptor of the peptide sequences and the Machine Learning technique for the best classifier. The best model represents a Random Forest classifier based on two features of the embedded and non-embedded graphs. The performance of the model is excellent, considering similar models in the field, with an Area Under the Receiver Operating Characteristic Curve (AUROC) value of 0.938 and true positive rate (TPR) of 0.886 (test subset). The prediction of new nucleotide binding peptides with this model could be useful for drug target studies in drug development. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification and characterization of polydimethylsiloxane-binding peptides (PDMS-tag) for oriented immobilization of functional protein on a PDMS surface.

PubMed

Kumada, Yoichi; Otsuki, Ryoko; Sakoda, Yumiko; Akai, Ryota; Matoba, Kazutaka; Katayama, Junko; Kishimoto, Michimasa; Horiuchi, Jun-Ichi

2016-10-20

In this study we focused on identifying and characterizing polydimethylsiloxane-binding peptides (PDMS-tags) that show a strong binding affinity towards a PDMS surface. Three kinds of E. coli host proteins (ELN, OMC and TPA) that were preferentially adsorbed onto a PDMS surface were identified from the E. coli cell lysate via 2-D electrophoresis and MALDI TOF MS. Digestion of these PDMS-binding proteins by 3 types of proteases (trypsin, chymotrypsin and V8 protease) resulted in the production of a wide variety of peptide fragments with different amino acid biases. Nine types of peptide fragments showing binding affinities to a PDMS surface were identified, and they were genetically fused at the C-terminal region of glutathione S-transferase (GST). The adsorption kinetics of peptide-fused GSTs to a PDMS surface were evaluated using a quartz crystal microbalance (QCM) sensor equipped with a sensor chip coated with a PDMS thin film. Consequently, all GSTs fused with the peptides adsorbed at a level higher than that of wild-type GST. In particular, the adsorption levels of GSTs fused with ELN-V81, TPA-V81, and OMC-V81 peptides were 8- to 10-fold higher than that of the wild-type GST. These results indicated that the selected peptides possessed a strong binding affinity towards a PDMS surface even in cases where they were introduced to the C-terminal region of a model protein. The remaining activities of GSTs with PDMS-binding peptides were also greater than that of the wild-type GST. Almost a third (30%) of enzymatic activity was maintained by genetic fusion of the peptide ELN-V81, compared with only 1.5% of wild-type GST in the adsorption state. Thus, the PDMS-binding peptides (PDMS-tags) identified in this study will be considerably useful for the site-specific immobilization of functional proteins to a PDMS surface, which will be a powerful tool in the fabrication of protein-based micro-reactors and biosearation chips. Copyright © 2016 Elsevier B.V. All rights

Delivering heparin-binding insulin-like growth factor 1 with self-assembling peptide hydrogels.

PubMed

Florine, Emily M; Miller, Rachel E; Liebesny, Paul H; Mroszczyk, Keri A; Lee, Richard T; Patwari, Parth; Grodzinsky, Alan J

2015-02-01

Heparin-binding insulin-like growth factor 1 (HB-IGF-1) is a fusion protein of IGF-1 with the HB domain of heparin-binding epidermal growth factor-like growth factor. A single dose of HB-IGF-1 has been shown to bind specifically to cartilage and to promote sustained upregulation of proteoglycan synthesis in cartilage explants. Achieving strong integration between native cartilage and tissue-engineered cartilage remains challenging. We hypothesize that if a growth factor delivered by the tissue engineering scaffold could stimulate enhanced matrix synthesis by both the cells within the scaffold and the adjacent native cartilage, integration could be enhanced. In this work, we investigated methods for adsorbing HB-IGF-1 to self-assembling peptide hydrogels to deliver the growth factor to encapsulated chondrocytes and cartilage explants cultured with growth factor-loaded hydrogels. We tested multiple methods for adsorbing HB-IGF-1 in self-assembling peptide hydrogels, including adsorption prior to peptide assembly, following peptide assembly, and with/without heparan sulfate (HS, a potential linker between peptide molecules and HB-IGF-1). We found that HB-IGF-1 and HS were retained in the peptide for all tested conditions. A subset of these conditions was then studied for their ability to stimulate increased matrix production by gel-encapsulated chondrocytes and by chondrocytes within adjacent native cartilage. Adsorbing HB-IGF-1 or IGF-1 prior to peptide assembly was found to stimulate increased sulfated glycosaminoglycan per DNA and hydroxyproline content of chondrocyte-seeded hydrogels compared with basal controls at day 10. Cartilage explants cultured adjacent to functionalized hydrogels had increased proteoglycan synthesis at day 10 when HB-IGF-1 was adsorbed, but not IGF-1. We conclude that delivery of HB-IGF-1 to focal defects in cartilage using self-assembling peptide hydrogels is a promising technique that could aid cartilage repair via enhanced matrix

Molecular Determinants of Peptide Binding to Two Common Rhesus Macaque Major Histocompatibility Complex Class II Molecules

PubMed Central

Dzuris, John L.; Sidney, John; Horton, Helen; Correa, Rose; Carter, Donald; Chesnut, Robert W.; Watkins, David I.; Sette, Alessandro

2001-01-01

Major histocompatibility complex class II molecules encoded by two common rhesus macaque alleles Mamu-DRB1*0406 and Mamu-DRB*w201 have been purified, and quantitative binding assays have been established. The structural requirements for peptide binding to each molecule were characterized by testing panels of single-substitution analogs of the two previously defined epitopes HIV Env242 (Mamu-DRB1*0406 restricted) and HIV Env482 (Mamu-DRB*w201 restricted). Anchor positions of both macaque DR molecules were spaced following a position 1 (P1), P4, P6, P7, and P9 pattern. The specific binding motif associated with each molecule was distinct, but largely overlapping, and was based on crucial roles of aromatic and/or hydrophobic residues at P1, P6, and P9. Based on these results, a tentative Mamu class II DR supermotif was defined. This pattern is remarkably similar to a previously defined human HLA-DR supermotif. Similarities in binding motifs between human HLA and macaque Mamu-DR molecules were further illustrated by testing a panel of more than 60 different single-substitution analogs of the HLA-DR-restricted HA 307–319 epitope for binding to Mamu-DRB*w201 and HLA-DRB1*0101. The Mamu-DRB1*0406 and -DRB*w201 binding capacity of a set of 311 overlapping peptides spanning the entire simian immunodeficiency virus (SIV) genome was also evaluated. Ten peptides capable of binding both molecules were identified, together with 19 DRB1*0406 and 43 DRB*w201 selective binders. The Mamu-DR supermotif was found to be present in about 75% of the good binders and in 50% of peptides binding with intermediate affinity but only in approximately 25% of the peptides which did not bind either Mamu class II molecule. Finally, using flow cytometric detection of antigen-induced intracellular gamma interferon, we identify a new CD4+ T-lymphocyte epitope encoded within the Rev protein of SIV. PMID:11602736

Human Leukocyte Antigen (HLA) B27 Allotype-Specific Binding and Candidate Arthritogenic Peptides Revealed through Heuristic Clustering of Data-independent Acquisition Mass Spectrometry (DIA-MS) Data*

PubMed Central

Schittenhelm, Ralf B.; Sivaneswaran, Saranjah; Lim Kam Sian, Terry C. C.; Croft, Nathan P.; Purcell, Anthony W.

2016-01-01

Expression of HLA-B27 is strongly associated with ankylosing spondylitis (AS) and other spondyloarthropathies. While this is true for the majority of HLA-B27 allotypes, HLA-B*27:06 and HLA-B*27:09 are not associated with AS. These two subtypes contain polymorphisms that are ideally positioned to influence the bound peptide repertoire. The existence of disease-inducing peptides (so-called arthritogenic peptides) has therefore been proposed that are exclusively presented by disease-associated HLA-B27 allotypes. However, we have recently demonstrated that this segregation of allotype-bound peptides is not the case and that many peptides that display sequence features predicted to favor binding to disease-associated subtypes are also capable of being presented naturally by protective alleles. To further probe more subtle quantitative changes in peptide presentation, we have used a combination of data-independent acquisition (DIA) and multiple reaction monitoring (MRM) mass spectrometry to quantify the abundance of 1646 HLA-B27 restricted peptides across the eight most frequent HLA-B27 allotypes (HLA-B*27:02-HLA-B*27:09). We utilized K means cluster analysis to group peptides with similar allelic binding preferences across the eight HLA-B27 allotypes, which enabled us to identify the most-stringent binding characteristics for each HLA-B27 allotype and further refined their existing consensus-binding motifs. Moreover, a thorough analysis of this quantitative dataset led to the identification of 26 peptides, which are presented in lower abundance by HLA-B*27:06 and HLA-B*27:09 compared with disease-associated HLA-B27 subtypes. Although these differences were observed to be very subtle, these 26 peptides might encompass the sought-after arthritogenic peptide(s). PMID:26929215

Human Leukocyte Antigen (HLA) B27 Allotype-Specific Binding and Candidate Arthritogenic Peptides Revealed through Heuristic Clustering of Data-independent Acquisition Mass Spectrometry (DIA-MS) Data.

PubMed

Schittenhelm, Ralf B; Sivaneswaran, Saranjah; Lim Kam Sian, Terry C C; Croft, Nathan P; Purcell, Anthony W

2016-06-01

Expression of HLA-B27 is strongly associated with ankylosing spondylitis (AS) and other spondyloarthropathies. While this is true for the majority of HLA-B27 allotypes, HLA-B*27:06 and HLA-B*27:09 are not associated with AS. These two subtypes contain polymorphisms that are ideally positioned to influence the bound peptide repertoire. The existence of disease-inducing peptides (so-called arthritogenic peptides) has therefore been proposed that are exclusively presented by disease-associated HLA-B27 allotypes. However, we have recently demonstrated that this segregation of allotype-bound peptides is not the case and that many peptides that display sequence features predicted to favor binding to disease-associated subtypes are also capable of being presented naturally by protective alleles. To further probe more subtle quantitative changes in peptide presentation, we have used a combination of data-independent acquisition (DIA) and multiple reaction monitoring (MRM) mass spectrometry to quantify the abundance of 1646 HLA-B27 restricted peptides across the eight most frequent HLA-B27 allotypes (HLA-B*27:02-HLA-B*27:09). We utilized K means cluster analysis to group peptides with similar allelic binding preferences across the eight HLA-B27 allotypes, which enabled us to identify the most-stringent binding characteristics for each HLA-B27 allotype and further refined their existing consensus-binding motifs. Moreover, a thorough analysis of this quantitative dataset led to the identification of 26 peptides, which are presented in lower abundance by HLA-B*27:06 and HLA-B*27:09 compared with disease-associated HLA-B27 subtypes. Although these differences were observed to be very subtle, these 26 peptides might encompass the sought-after arthritogenic peptide(s). © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies

DOE PAGES

Donaldson, Joshua M.; Zer, Cindy; Avery, Kendra N.; ...

2013-10-07

Capitalizing on their extraordinary specificity, monoclonal antibodies (mAbs) have become one of the most reengineered classes of biological molecules. A major goal in many of these engineering efforts is to add new functionality to the parental mAb, including the addition of cytotoxins and imaging agents for medical applications. Herein, we present a unique peptide-binding site within the central cavity of the fragment antigen binding framework region of the chimeric, anti-epidermal growth factor receptor mAb cetuximab. We demonstrate through diffraction methods, biophysical studies, and sequence analysis that this peptide, a meditope, has moderate affinity for the Fab, is specific to cetuximabmore » (i.e., does not bind to human IgGs), and has no significant effect on antigen binding. We further demonstrate by diffraction studies and biophysical methods that the meditope binding site can be grafted onto the anti-human epidermal growth factor receptor 2 mAb trastuzumab, and that the antigen binding affinity of the grafted trastuzumab is indistinguishable from the parental mAb. Lastly, we demonstrate a bivalent meditope variant binds specifically and stably to antigen-bearing cells only in the presence of the meditope-enabled mAbs. Collectively, this finding and the subsequent characterization and engineering efforts indicate that this unique interface could serve as a noncovalent “linker” for any meditope-enabled mAb with applications in multiple mAb-based technologies including diagnostics, imaging, and therapeutic delivery.« less

Specific labeling of the thyroxine binding site in thyroxine-binding globulin: determination of the amino acid composition of a labeled peptide fragment isolated from a proteolytic digest of the derivatized protein.

PubMed

Tabachnick, M; Perret, V

1987-08-01

[125I] Thyroxine has been covalently bound to the thyroxine binding site in thyroxine-binding globulin by reaction with the bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. An average of 0.47 mol of [125I] thyroxine was incorporated per mol protein; nonspecific binding amounted to 8%. A labeled peptide fragment was isolated from a proteolytic digest of the derivatized protein by HPLC and its amino acid composition was determined. Comparison with the amino acid sequence of thyroxine-binding globulin indicated partial correspondence of the labeled peptide with two possible regions in the protein. These regions also coincide with part of the barrel structure present in the closely homologous protein, alpha 1-antitrypsin.

Analysis of the (Trimethylsilyl)propionic Acid-β(12-28) Peptide Binding Equilibrium with NMR Spectroscopy.

PubMed

Jayawickrama, D A; Larive, C K

1999-06-01

The binding of a small molecule, (trimethylsilyl)propionic acid (TSP), to a 17-residue peptide, β(12-28), is examined using (1)H NMR spectroscopy. β(12-28) (VHHQKLVFFAEDVGSNK) is a central fragment of the 40-42-residue Alzheimer's-associated Aβ peptide. This peptide has been previously shown to form soluble aggregates in low-pH aqueous solution. The TSP resonance is broadened appreciably in solutions containing relatively high concentrations (∼2 mM) of the peptide. The changes in TSP line width measured by titration of a peptide solution with TSP indicate a 1:1 binding stoichiometry. If the concentrations of both the peptide and TSP are reduced by 1 order of magnitude, the resonances of both species are sharp, suggesting that TSP binds predominately to the aggregated peptide. Nuclear Overhauser effect experiments indicate that the TSP interacts predominately with the side chains of the aliphatic peptide residues Leu(17) and Val(18). Pulsed-field gradient NMR measurements of TSP and peptide diffusion coefficients provide a more quantitative picture of the TSP-peptide binding equilibrium. The measured diffusion coefficients were used to calculate the fractions of the free and bound TSP. These results substantiate the conclusion that the stoichiometry of the TSP-peptide binding equilibrium is essentially 1:1 and further indicate anticooperative behavior in solutions containing an excess of TSP resulting in a dissociation of the peptide aggregates.

Peptide functionalized gold nanoparticles: the influence of pH on binding efficiency

NASA Astrophysics Data System (ADS)

Harrison, Emma; Hamilton, Jeremy W. J.; Macias-Montero, Manuel; Dixon, Dorian

2017-07-01

We report herein on the synthesis of mixed monolayer gold nanoparticles (AuNPs) capped with both polyethylene glycol (PEG) and one of three peptides. Either a receptor-mediated endocytosis peptide, an endosomal escape pathway (H5WYG) peptide or the Nrp-1 targeting RGD peptide (CRGDK) labeled with FITC. All three peptides have a thiol containing cysteine residue which can be used to bind the peptides to the AuNPs. In order to investigate the influence of pH on peptide attachment, PEGylated AuNPs were centrifuged, the supernatant removed, and the nanoparticles were then re-suspended in a range of pH buffer solutions above, below and at the respective isoelectric points of the peptides before co-functionalization. Peptide attachment was investigated using dynamic light scattering, Ultra-violet visible spectroscopy (UV/Vis), FTIR and photo luminescence spectroscopy. UV/Vis analysis coupled with protein assay results and photoluminescence of the FITC tagged RGD peptide concluded that a pH of ∼8 optimized the cysteine binding and stability, irrespective of the peptide used.

Modeling alternative binding registers of a minimal immunogenic peptide on two class II major histocompatibility complex (MHC II) molecules predicts polarized T-cell receptor (TCR) contact positions.

PubMed

Murray, J S; Fois, S D S; Schountz, T; Ford, S R; Tawde, M D; Brown, J C; Siahaan, T J

2002-03-01

Several major histocompatibility complex class II (MHC II) complexes with known minimal immunogenic peptides have now been solved by X-ray crystallography. Specificity pockets within the MHC II binding groove provide distinct peptide contacts that influence peptide conformation and define the binding register within different allelic MHC II molecules. Altering peptide ligands with respect to the residues that contact the T-cell receptor (TCR) can drastically change the nature of the ensuing immune response. Here, we provide an example of how MHC II (I-A) molecules may indirectly effect TCR contacts with a peptide and drive functionally distinct immune responses. We modeled the same immunogenic 12-amino acid peptide into the binding grooves of two allelic MHC II molecules linked to distinct cytokine responses against the peptide. Surprisingly, the favored conformation of the peptide in each molecule was distinct with respect to the exposure of the N- or C-terminus of the peptide above the MHC II binding groove. T-cell clones derived from each allelic MHC II genotype were found to be allele-restricted with respect to the recognition of these N- vs. C-terminal residues on the bound peptide. Taken together, these data suggest that MHC II alleles may influence T-cell functions by restricting TCR access to specific residues of the I-A-bound peptide. Thus, these data are of significance to diseases that display genetic linkage to specific MHC II alleles, e.g. type 1 diabetes and rheumatoid arthritis.

Entropy-driven binding of opioid peptides induces a large domain motion in human dipeptidyl peptidase III

PubMed Central

Bezerra, Gustavo A.; Dobrovetsky, Elena; Viertlmayr, Roland; Dong, Aiping; Binter, Alexandra; Abramić, Marija; Macheroux, Peter; Dhe-Paganon, Sirano; Gruber, Karl

2012-01-01

Opioid peptides are involved in various essential physiological processes, most notably nociception. Dipeptidyl peptidase III (DPP III) is one of the most important enkephalin-degrading enzymes associated with the mammalian pain modulatory system. Here we describe the X-ray structures of human DPP III and its complex with the opioid peptide tynorphin, which rationalize the enzyme's substrate specificity and reveal an exceptionally large domain motion upon ligand binding. Microcalorimetric analyses point at an entropy-dominated process, with the release of water molecules from the binding cleft (“entropy reservoir”) as the major thermodynamic driving force. Our results provide the basis for the design of specific inhibitors that enable the elucidation of the exact role of DPP III and the exploration of its potential as a target of pain intervention strategies. PMID:22493238

Mutation Induced Conformational Change In CaMKII Peptide Alters Binding Affinity to CaM Through Alternate Binding Site

NASA Astrophysics Data System (ADS)

Ezerski, Jacob; Cheung, Margaret

CaM forms distinct conformation states through modifications in its charge distribution upon binding to Ca2+ ions. The occurrence of protein structural change resulting from an altered charge distribution is paramount in the scheme of cellular signaling. Not only is charge induced structural change observed in CaM, it is also seen in an essential binding target: calmodulin-depended protein kinase II (CaMKII). In order to investigate the mechanism of selectivity in relation to changes in secondary structure, the CaM binding domain of CaMKII is isolated. Experimentally, charged residues of the CaMKII peptide are systematically mutated to alanine, resulting in altered binding kinetics between the peptide and the Ca2+ saturated state of CaM. We perform an all atom simulation of the wildtype (RRK) and mutated (AAA) CaMKII peptides and generate structures from the trajectory. We analyze RRK and AAA using DSSP and find significant structural differences due to the mutation. Structures from the RRK and AAA ensembles are then selected and docked onto the crystal structure of Ca2+ saturated CaM. We observe that RRK binds to CaM at the C-terminus, whereas the 3-residue mutation, AAA, shows increased patterns of binding to the N-terminus and linker regions of CaM. Due to the conformational change of the peptide ensemble from charged residue mutation, a distinct change in the binding site can be seen, which offers an explanation to experimentally observed changes in kinetic binding rates

Selective Intracellular Delivery of Ganglioside GM3-Binding Peptide through Caveolae/Raft-Mediated Endocytosis.

PubMed

Matsubara, Teruhiko; Otani, Ryohei; Yamashita, Miki; Maeno, Haruka; Nodono, Hanae; Sato, Toshinori

2017-02-13

Glycosphingolipids are major components of the membrane raft, and several kinds of viruses and bacterial toxins are known to bind to glycosphingolipids in the membrane raft. Since the viral genes and pathogenic proteins that are taken into cells are directly delivered to their target organelles, caveolae/raft-mediated endocytosis represents a promising pathway for specific delivery. In the present study, we demonstrated the ability of an artificial pentadecapeptide, which binds to ganglioside GM3, to deliver protein into cells by caveolae/raft-mediated endocytosis. The cellular uptake of a biotinylated GM3-binding peptide (GM3BP)-avidin complex into HeLa cells was observed, and the cellular uptake of this complex was inhibited by an incubation with sialic acid or endocytic inhibitors such as methyl-ß-cyclodextrin, and also by an incubation at 4 °C. These results indicate that the GM3BP-avidin complex bind to GM3 in membrane raft, and are taken into cell through caveolae/raft-mediated endocytosis. The GM3BP-avidin complex was transported into cells and localized around the nucleus more slowly than a human immunodeficiency virus type 1 TAT peptide. Furthermore, the uptake of a green fluorescent protein (GFP) linked with GM3BP into HeLa cells was similar to that of the GM3BP-avidin complex, and the localization of the GM3BP-GFP fusion protein was markedly different with that of the TAT-GFP fusion protein. The uptake and trafficking of GM3BP were distinguished from conventional cell-penetrating peptides. GM3BP has potential as a novel peptide for the selective delivery of therapeutic proteins and materials into cells in addition to being a cell-penetrating peptide.

Rapid PD-L1 detection in tumors with PET using a highly specific peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chatterjee, Samit; Lesniak, Wojciech G.; Miller, Michelle S.

Molecular imaging can report on the status of the tumor immune microenvironment and guide immunotherapeutic strategies to enhance the efficacy of immune modulation therapies. Imaging agents that can rapidly report on targets of immunomodulatory therapies are few. The programmed death ligand 1 (PD-L1) is an immune checkpoint protein over-expressed in several cancers and contributes to tumor immune suppression. Tumor PD-L1 expression is indicative of tumor response to PD-1 and PD-L1 targeted therapies. Herein, we report a highly specific peptide-based positron emission tomography (PET) imaging agent for PD-L1. We assessed the binding modes of the peptide WL12 to PD-L1 by dockingmore » studies, developed a copper-64 labeled WL12 ([{sup 64}Cu]WL12), and performed its evaluation in vitro, and in vivo by PET imaging, biodistribution and blocking studies. Our results show that [{sup 64}Cu]WL12 can be used to detect tumor PD-L1 expression specifically and soon after injection of the radiotracer, to fit within the standard clinical workflow of imaging within 60 min of administration. - Highlights: • A highly specific PD-L1 binding peptide, WL12, was developed as a PET imaging agent. • [{sup 64}Cu]WL12 demonstrates specific binding to PD-L1 in vitro and in vivo. • [{sup 64}Cu]WL12-PET allows PD-L1 detection in cancers within 60 min of administration. • WL12 binding interactions with PD-L1 overlaps with that of PD-1.« less

Zinc(II) binds to the neuroprotective peptide humanin.

PubMed

Armas, Ambar; Sonois, Vanessa; Mothes, Emmanuelle; Mazarguil, Honoré; Faller, Peter

2006-10-01

The abnormal accumulation of the peptide amyloid-beta in the form of senile (or amyloid) plaques is one of the hallmarks of Alzheimer's disease (AD). Zinc ions have been implicated in AD and plaques formation. Recently, the peptide humanin has been discovered. Humanin showed neuroprotective activity against amyloid-beta insults. Here the question investigated is if humanin could interact directly with Zn(II). It is shown that Zn(II) and its substitutes Cd(II)/Co(II) bind to humanin via a thiolate bond from the side chain of the single cysteine at position 8. The low intensity of the d-d bands of Co(II)-humanin indicated an octahedral coordination geometry. Titration experiments suggest that Zn(II) binds to humanin with an apparent affinity in the low muM range. This apparent Zn-binding affinity is in the same order as for amyloid-beta and glutathione and could thus be of physiological relevance.

Computational Exploration of a Protein Receptor Binding Space with Student Proposed Peptide Ligands

PubMed Central

King, Matthew D.; Phillips, Paul; Turner, Matthew W.; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; Mcdougal, Owen M.

2017-01-01

Computational molecular docking is a fast and effective in silico method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The DockoMatic tutorial described herein provides a framework by which instructors can guide students through a drug screening exercise. Using receptor models derived from readily available protein crystal structures, docking programs have the ability to predict ligand binding properties, such as preferential binding orientations and binding affinities. The use of computational studies can significantly enhance complimentary wet chemical experimentation by providing insight into the important molecular interactions within the system of interest, as well as guide the design of new candidate ligands based on observed binding motifs and energetics. In this laboratory tutorial, the graphical user interface, DockoMatic, facilitates docking job submissions to the docking engine, AutoDock 4.2. The purpose of this exercise is to successfully dock a 17-amino acid peptide, α-conotoxin TxIA, to the acetylcholine binding protein from Aplysia californica-AChBP to determine the most stable binding configuration. Each student will then propose two specific amino acid substitutions of α-conotoxin TxIA to enhance peptide binding affinity, create the mutant in DockoMatic, and perform docking calculations to compare their results with the class. Students will also compare intermolecular forces, binding energy, and geometric orientation of their prepared analog to their initial α-conotoxin TxIA docking results. PMID:26537635

Development of a Novel Tetravalent Synthetic Peptide That Binds to Phosphatidic Acid.

PubMed

Ogawa, Rina; Nagao, Kohjiro; Taniuchi, Kentaro; Tsuchiya, Masaki; Kato, Utako; Hara, Yuji; Inaba, Takehiko; Kobayashi, Toshihide; Sasaki, Yoshihiro; Akiyoshi, Kazunari; Watanabe-Takahashi, Miho; Nishikawa, Kiyotaka; Umeda, Masato

2015-01-01

We employed a multivalent peptide-library screening technique to identify a peptide motif that binds to phosphatidic acid (PA), but not to other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). A tetravalent peptide with the sequence motif of MARWHRHHH, designated as PAB-TP (phosphatidic acid-binding tetravalent peptide), was shown to bind as low as 1 mol% of PA in the bilayer membrane composed of PC and cholesterol. Kinetic analysis of the interaction between PAB-TP and the membranes containing 10 mol% of PA showed that PAB-TP associated with PA with a low dissociation constant of KD = 38 ± 5 nM. Coexistence of cholesterol or PE with PA in the membrane enhanced the PAB-TP binding to PA by increasing the ionization of the phosphomonoester head group as well as by changing the microenvironment of PA molecules in the membrane. Amino acid replacement analysis demonstrated that the tryptophan residue at position 4 of PAB-TP was involved in the interaction with PA. Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA. Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane. The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

Hydroxyapatite-binding peptides for bone growth and inhibition

DOEpatents

Bertozzi, Carolyn R [Berkeley, CA; Song, Jie [Shrewsbury, MA; Lee, Seung-Wuk [Walnut Creek, CA

2011-09-20

Hydroxyapatite (HA)-binding peptides are selected using combinatorial phage library display. Pseudo-repetitive consensus amino acid sequences possessing periodic hydroxyl side chains in every two or three amino acid sequences are obtained. These sequences resemble the (Gly-Pro-Hyp).sub.x repeat of human type I collagen, a major component of extracellular matrices of natural bone. A consistent presence of basic amino acid residues is also observed. The peptides are synthesized by the solid-phase synthetic method and then used for template-driven HA-mineralization. Microscopy reveal that the peptides template the growth of polycrystalline HA crystals .about.40 nm in size.

Activation of erythropoietin receptor in the absence of hormone by a peptide that binds to a domain different from the hormone binding site

PubMed Central

Naranda, Tatjana; Wong, Kenneth; Kaufman, R. Ilene; Goldstein, Avram; Olsson, Lennart

1999-01-01

Applying a homology search method previously described, we identified a sequence in the extracellular dimerization site of the erythropoietin receptor, distant from the hormone binding site. A peptide identical to that sequence was synthesized. Remarkably, it activated receptor signaling in the absence of erythropoietin. Neither the peptide nor the hormone altered the affinity of the other for the receptor; thus, the peptide does not bind to the hormone binding site. The combined activation of signal transduction by hormone and peptide was strongly synergistic. In mice, the peptide acted like the hormone, protecting against the decrease in hematocrit caused by carboplatin. PMID:10377456

In silico peptide-binding predictions of passerine MHC class I reveal similarities across distantly related species, suggesting convergence on the level of protein function.

PubMed

Follin, Elna; Karlsson, Maria; Lundegaard, Claus; Nielsen, Morten; Wallin, Stefan; Paulsson, Kajsa; Westerdahl, Helena

2013-04-01

The major histocompatibility complex (MHC) genes are the most polymorphic genes found in the vertebrate genome, and they encode proteins that play an essential role in the adaptive immune response. Many songbirds (passerines) have been shown to have a large number of transcribed MHC class I genes compared to most mammals. To elucidate the reason for this large number of genes, we compared 14 MHC class I alleles (α1-α3 domains), from great reed warbler, house sparrow and tree sparrow, via phylogenetic analysis, homology modelling and in silico peptide-binding predictions to investigate their functional and genetic relationships. We found more pronounced clustering of the MHC class I allomorphs (allele specific proteins) in regards to their function (peptide-binding specificities) compared to their genetic relationships (amino acid sequences), indicating that the high number of alleles is of functional significance. The MHC class I allomorphs from house sparrow and tree sparrow, species that diverged 10 million years ago (MYA), had overlapping peptide-binding specificities, and these similarities across species were also confirmed in phylogenetic analyses based on amino acid sequences. Notably, there were also overlapping peptide-binding specificities in the allomorphs from house sparrow and great reed warbler, although these species diverged 30 MYA. This overlap was not found in a tree based on amino acid sequences. Our interpretation is that convergent evolution on the level of the protein function, possibly driven by selection from shared pathogens, has resulted in allomorphs with similar peptide-binding repertoires, although trans-species evolution in combination with gene conversion cannot be ruled out.

Side-chain conformational space analysis (SCSA): A multi conformation-based QSAR approach for modeling and prediction of protein-peptide binding affinities

NASA Astrophysics Data System (ADS)

Zhou, Peng; Chen, Xiang; Shang, Zhicai

2009-03-01

In this article, the concept of multi conformation-based quantitative structure-activity relationship (MCB-QSAR) is proposed, and based upon that, we describe a new approach called the side-chain conformational space analysis (SCSA) to model and predict protein-peptide binding affinities. In SCSA, multi-conformations (rather than traditional single-conformation) have received much attention, and the statistical average information on multi-conformations of side chains is determined using self-consistent mean field theory based upon side chain rotamer library. Thereby, enthalpy contributions (including electrostatic, steric, hydrophobic interaction and hydrogen bond) and conformational entropy effects to the binding are investigated in terms of occurrence probability of residue rotamers. Then, SCSA was applied into the dataset of 419 HLA-A*0201 binding peptides, and nonbonding contributions of each position in peptide ligands are well determined. For the peptides, the hydrogen bond and electrostatic interactions of the two ends are essential to the binding specificity, van der Waals and hydrophobic interactions of all the positions ensure strong binding affinity, and the loss of conformational entropy at anchor positions partially counteracts other favorable nonbonding effects.

Improved methods for predicting peptide binding affinity to MHC class II molecules.

PubMed

Jensen, Kamilla Kjaergaard; Andreatta, Massimo; Marcatili, Paolo; Buus, Søren; Greenbaum, Jason A; Yan, Zhen; Sette, Alessandro; Peters, Bjoern; Nielsen, Morten

2018-07-01

Major histocompatibility complex class II (MHC-II) molecules are expressed on the surface of professional antigen-presenting cells where they display peptides to T helper cells, which orchestrate the onset and outcome of many host immune responses. Understanding which peptides will be presented by the MHC-II molecule is therefore important for understanding the activation of T helper cells and can be used to identify T-cell epitopes. We here present updated versions of two MHC-II-peptide binding affinity prediction methods, NetMHCII and NetMHCIIpan. These were constructed using an extended data set of quantitative MHC-peptide binding affinity data obtained from the Immune Epitope Database covering HLA-DR, HLA-DQ, HLA-DP and H-2 mouse molecules. We show that training with this extended data set improved the performance for peptide binding predictions for both methods. Both methods are publicly available at www.cbs.dtu.dk/services/NetMHCII-2.3 and www.cbs.dtu.dk/services/NetMHCIIpan-3.2. © 2018 John Wiley & Sons Ltd.

Molecular specialization of breast vasculature: A breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature

NASA Astrophysics Data System (ADS)

Essler, Markus; Ruoslahti, Erkki

2002-02-01

In vivo phage display identifies peptides that selectively home to the vasculature of individual organs, tissues, and tumors. Here we report the identification of a cyclic nonapeptide, CPGPEGAGC, which homes to normal breast tissue with a 100-fold selectivity over nontargeted phage. The homing of the phage is inhibited by its cognate synthetic peptide. Phage localization in tissue sections showed that the breast-homing phage binds to the blood vessels in the breast, but not in other tissues. The phage also bound to the vasculature of hyperplastic and malignant lesions in transgenic breast cancer mice. Expression cloning with a phage-displayed cDNA library yielded a phage that specifically bound to the breast-homing peptide. The cDNA insert was homologous to a fragment of aminopeptidase P. The homing peptide bound aminopeptidase P from malignant breast tissue in affinity chromatography. Antibodies against aminopeptidase P inhibited the in vitro binding of the phage-displayed cDNA to the peptide and the in vivo homing of phage carrying the peptide. These results indicate that aminopeptidase P is the receptor for the breast-homing peptide. This peptide may be useful in designing drugs for the prevention and treatment of breast cancer.

An interleukin 13 receptor α 2–specific peptide homes to human Glioblastoma multiforme xenografts

PubMed Central

Pandya, Hetal; Gibo, Denise M.; Garg, Shivank; Kridel, Steven; Debinski, Waldemar

2012-01-01

Interleukin 13 receptor α 2 (IL-13Rα2) is a glioblastoma multiforme (GBM)–associated plasma membrane receptor, a brain tumor of dismal prognosis. Here, we isolated peptide ligands for IL-13Rα2 with use of a cyclic disulphide-constrained heptapeptide phages display library and 2 in vitro biopanning schemes with GBM cells that do (G26-H2 and SnB19-pcDNA cells) or do not (G26-V2 and SnB19-asIL-13Rα2 cells) over-express IL-13Rα2. We identified 3 peptide phages that bind to IL-13Rα2 in cellular and protein assays. One of the 3 peptide phages, termed Pep-1, bound to IL-13Rα2 with the highest specificity, surprisingly, also in a reducing environment. Pep-1 was thus synthesized and further analyzed in both linear and disulphide-constrained forms. The linear peptide bound to IL-13Rα2 more avidly than did the disulphide-constrained form and was efficiently internalized by IL-13Rα2–expressing GBM cells. The native ligand, IL-13, did not compete for the Pep-1 binding to the receptor and vice versa in any of the assays, indicating that the peptide might be binding to a site on the receptor different from the native ligand. Furthermore, we demonstrated by noninvasive near infrared fluorescence imaging in nude mice that Pep-1 binds and homes to both subcutaneous and orthotopic human GBM xenografts expressing IL-13Rα2 when injected by an intravenous route. Thus, we identified a linear heptapeptide specific for the IL-13Rα2 that is capable of crossing the blood-brain tumor barrier and homing to tumors. Pep-1 can be further developed for various applications in cancer and/or inflammatory diseases. PMID:21946118

Phage-display screening identifies LMP1-binding peptides targeting the C-terminus region of the EBV oncoprotein.

PubMed

Ammous-Boukhris, Nihel; Mosbah, Amor; Sahli, Emna; Ayadi, Wajdi; Hadhri-Guiga, Boutheina; Chérif, Ameur; Gargouri, Ali; Mokdad-Gargouri, Raja

2016-11-01

Latent membrane protein 1 (LMP1), a major oncoprotein of Epstein Barr Virus (EBV) is responsible for transforming B lymphocytes in vitro. LMP1 is overexpressed in several EBV-associated malignancies, and different approaches have been developed to reduce its level and accordingly its oncogenic function in tumor tissues. This study aimed to use phage display peptide library to obtain peptides which could specifically bind to the cytoplasmic region of LMP1 to prevent its interaction with signaling proteins. The LMP1 C-terminus region was produced in bacterial E. coli and used as target for the phage library panning. After 3 rounds, 20 phage clones were randomly selected and 8 showed high binding affinity to the recombinant C-terminus LMP1 protein. The most interesting candidates are the FO5 "QPTKDSSPPLRV" and NO4 "STTSPPAVPHNN" peptides since both bind the C-terminus LMP1 as showed by molecular docking. Furthermore, sequence alignment revealed that the FO5 peptide shared sequence similarity with the Death Receptor 4 which belongs to the tumor necrosis factor-related apoptosis-inducing receptor which plays key role in anti-tumor immunity. Copyright © 2016 Elsevier Inc. All rights reserved.

Cyclic Peptidic Mimetics of Apollo Peptides Targeting Telomeric Repeat Binding Factor 2 (TRF2) and Apollo Interaction.

PubMed

Chen, Xia; Liu, Liu; Chen, Yong; Yang, Yuting; Yang, Chao-Yie; Guo, Tianyue; Lei, Ming; Sun, Haiying; Wang, Shaomeng

2018-05-10

Telomeric repeat binding factor 2 (TRF2) is a telomere-associated protein that plays an important role in the formation of the 3' single strand DNA overhang and the "T loop", two structures critical for the stability of the telomeres. Apollo is a 5'-exonuclease recruited by TRF2 to the telomere and contributes to the formation of the 3' single strand DNA overhang. Knocking down of Apollo can induce DNA damage response similar to that caused by the knocking down of TRF2. In this Letter, we report the design and synthesis of a class of cyclic peptidic mimetics of the TRFH binding motif of Apollo (Apollo TBM ). We found conformational control of the C terminal residues of Apollo TBM can effectively improve the binding affinity. We have obtained a crystal structure of a cyclic peptidic Apollo peptide mimetic ( 34 ) complexed with TRF2, which provides valuable guidance to the future design of TRF2 inhibitors.

Computational exploration of a protein receptor binding space with student proposed peptide ligands.

PubMed

King, Matthew D; Phillips, Paul; Turner, Matthew W; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M

2016-01-01

Computational molecular docking is a fast and effective in silico method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The DockoMatic tutorial described herein provides a framework by which instructors can guide students through a drug screening exercise. Using receptor models derived from readily available protein crystal structures, docking programs have the ability to predict ligand binding properties, such as preferential binding orientations and binding affinities. The use of computational studies can significantly enhance complimentary wet chemical experimentation by providing insight into the important molecular interactions within the system of interest, as well as guide the design of new candidate ligands based on observed binding motifs and energetics. In this laboratory tutorial, the graphical user interface, DockoMatic, facilitates docking job submissions to the docking engine, AutoDock 4.2. The purpose of this exercise is to successfully dock a 17-amino acid peptide, α-conotoxin TxIA, to the acetylcholine binding protein from Aplysia californica-AChBP to determine the most stable binding configuration. Each student will then propose two specific amino acid substitutions of α-conotoxin TxIA to enhance peptide binding affinity, create the mutant in DockoMatic, and perform docking calculations to compare their results with the class. Students will also compare intermolecular forces, binding energy, and geometric orientation of their prepared analog to their initial α-conotoxin TxIA docking results. © 2015 The International Union of Biochemistry and Molecular Biology.

Quantitative modeling of peptide binding to TAP using support vector machine.

PubMed

Diez-Rivero, Carmen M; Chenlo, Bernardo; Zuluaga, Pilar; Reche, Pedro A

2010-01-01

The transport of peptides to the endoplasmic reticulum by the transporter associated with antigen processing (TAP) is a necessary step towards determining CD8 T cell epitopes. In this work, we have studied the predictive performance of support vector machine models trained on single residue positions and residue combinations drawn from a large dataset consisting of 613 nonamer peptides of known affinity to TAP. Predictive performance of these TAP affinity models was evaluated under 10-fold cross-validation experiments and measured using Pearson's correlation coefficients (R(p)). Our results show that every peptide position (P1-P9) contributes to TAP binding (minimum R(p) of 0.26 +/- 0.11 was achieved by a model trained on the P6 residue), although the largest contributions to binding correspond to the C-terminal end (R(p) = 0.68 +/- 0.06) and the P1 (R(p) = 0.51 +/- 0.09) and P2 (0.57 +/- 0.08) residues of the peptide. Training the models on additional peptide residues generally improved their predictive performance and a maximum correlation (R(p) = 0.89 +/- 0.03) was achieved by a model trained on the full-length sequences or a residue selection consisting of the first 5 N- and last 3 C-terminal residues of the peptides included in the training set. A system for predicting the binding affinity of peptides to TAP using the methods described here is readily available for free public use at http://imed.med.ucm.es/Tools/tapreg/. (c) 2009 Wiley-Liss, Inc.

Computational design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1 protease

PubMed Central

Altman, Michael D.; Nalivaika, Ellen A.; Prabu-Jeyabalan, Moses; Schiffer, Celia A.; Tidor, Bruce

2009-01-01

Drug resistance in HIV-1 protease, a barrier to effective treatment, is generally caused by mutations in the enzyme that disrupt inhibitor binding but still allow for substrate processing. Structural studies with mutant, inactive enzyme, have provided detailed information regarding how the substrates bind to the protease yet avoid resistance mutations; insights obtained inform the development of next generation therapeutics. Although structures have been obtained of complexes between substrate peptide and inactivated (D25N) protease, thermodynamic studies of peptide binding have been challenging due to low affinity. Peptides that bind tighter to the inactivated protease than the natural substrates would be valuable for thermodynamic studies as well as to explore whether the structural envelope observed for substrate peptides is a function of weak binding. Here, two computational methods — namely, charge optimization and protein design — were applied to identify peptide sequences predicted to have higher binding affinity to the inactivated protease, starting from an RT–RH derived substrate peptide. Of the candidate designed peptides, three were tested for binding with isothermal titration calorimetry, with one, containing a single threonine to valine substitution, measured to have more than a ten-fold improvement over the tightest binding natural substrate. Crystal structures were also obtained for the same three designed peptide complexes; they show good agreement with computational prediction. Thermodynamic studies show that binding is entropically driven, more so for designed affinity enhanced variants than for the starting substrate. Structural studies show strong similarities between natural and tighter-binding designed peptide complexes, which may have implications in understanding the molecular mechanisms of drug resistance in HIV-1 protease. PMID:17729291

[Planar molecular arrangements aid the design of MHC class II binding peptides].

PubMed

Cortés, A; Coral, J; McLachlan, C; Benítez, R; Pinilla, L

2017-01-01

The coupling between peptides and MHC-II proteins in the human immune system is not well understood. This work presents an evidence-based hypothesis of a guiding intermolecular force present in every human MHC-II protein (HLA-II). Previously, we examined the spatial positions of the fully conserved residues in all HLA-II protein types. In each one, constant planar patterns were revealed. These molecular planes comprise of amino acid groups of the same chemical species (for example, Gly) distributed across the protein structure. Each amino acid plane has a unique direction and this directional element offers spatial selectivity. Constant within all planes, too, is the presence of an aromatic residue possessing electrons in movement, leading the authors to consider that the planes generate electromagnetic fields that could serve as an attractive force in a single direction. Selection and attraction between HLA-II molecules and antigen peptides would, therefore, be non-random, resulting in a coupling mechanism as effective and rapid as is clearly required in the immune response. On the basis of planar projections onto the HLA-II groove, modifications were made by substituting the key residues in the class II-associated invariant chain peptide-a peptide with a universal binding affinity-resulting in eight different modified peptides with affinities greater than that of the unmodified peptide. Accurate and reliable prediction of MHC class II-binding peptides may facilitate the design of universal vaccine-peptides with greatly enhanced binding affinities. The proposed mechanisms of selection, attraction and coupling between HLA-II and antigen peptides are explained further in the paper.

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

PubMed Central

Yahashiri, Atsushi; Jorgenson, Matthew A.; Weiss, David S.

2015-01-01

Bacterial SPOR domains bind peptidoglycan (PG) and are thought to target proteins to the cell division site by binding to “denuded” glycan strands that lack stem peptides, but uncertainties remain, in part because septal-specific binding has yet to be studied in a purified system. Here we show that fusions of GFP to SPOR domains from the Escherichia coli cell-division proteins DamX, DedD, FtsN, and RlpA all localize to septal regions of purified PG sacculi obtained from E. coli and Bacillus subtilis. Treatment of sacculi with an amidase that removes stem peptides enhanced SPOR domain binding, whereas treatment with a lytic transglycosylase that removes denuded glycans reduced SPOR domain binding. These findings demonstrate unequivocally that SPOR domains localize by binding to septal PG, that the physiologically relevant binding site is indeed a denuded glycan, and that denuded glycans are enriched in septal PG rather than distributed uniformly around the sacculus. Accumulation of denuded glycans in the septal PG of both E. coli and B. subtilis, organisms separated by 1 billion years of evolution, suggests that sequential removal of stem peptides followed by degradation of the glycan backbone is an ancient feature of PG turnover during bacterial cell division. Linking SPOR domain localization to the abundance of a structure (denuded glycans) present only transiently during biogenesis of septal PG provides a mechanism for coordinating the function of SPOR domain proteins with the progress of cell division. PMID:26305949

Tissue-specific effects of peptides.

PubMed

Khavinson, V K

2001-08-01

Synthetic peptides (cytogens) Cortagen, Epithalon, Livagen, and Vilon stimulated the growth of explants from rat brain cortex, subcortical structures, liver, and thymus, respectively, in organotypic cultures. These peptides produced tissue-specific effects: they stimulated the growth of explants from tissues, whose cytomedins (peptide complexes) were used for chemical synthesis.

Identification of Four-Jointed Box 1 (FJX1)-Specific Peptides for Immunotherapy of Nasopharyngeal Carcinoma.

PubMed

Chai, San Jiun; Yap, Yoke Yeow; Foo, Yoke Ching; Yap, Lee Fah; Ponniah, Sathibalan; Teo, Soo Hwang; Cheong, Sok Ching; Patel, Vyomesh; Lim, Kue Peng

2015-01-01

Nasopharyngeal carcinoma (NPC) is highly prevalent in South East Asia and China. The poor outcome is due to late presentation, recurrence, distant metastasis and limited therapeutic options. For improved treatment outcome, immunotherapeutic approaches focusing on dendritic and autologous cytotoxic T-cell based therapies have been developed, but cost and infrastructure remain barriers for implementing these in low-resource settings. As our prior observations had found that four-jointed box 1 (FJX1), a tumor antigen, is overexpressed in NPCs, we investigated if short 9-20 amino acid sequence specific peptides matching to FJX1 requiring only intramuscular immunization to train host immune systems would be a better treatment option for this disease. Thus, we designed 8 FJX1-specific peptides and implemented an assay system to first, assess the binding of these peptides to HLA-A2 molecules on T2 cells. After, ELISPOT assays were used to determine the peptides immunogenicity and ability to induce potential cytotoxicity activity towards cancer cells. Also, T-cell proliferation assay was used to evaluate the potential of MHC class II peptides to stimulate the expansion of isolated T-cells. Our results demonstrate that these peptides are immunogenic and peptide stimulated T-cells were able to induce peptide-specific cytolytic activity specifically against FJX1-expressing cancer cells. In addition, we demonstrated that the MHC class II peptides were capable of inducing T-cell proliferation. Our results suggest that these peptides are capable of inducing specific cytotoxic cytokines secretion against FJX1-expressing cancer cells and serve as a potential vaccine-based therapy for NPC patients.

Rational Design of Peptide-Functionalized Surface Plasmon Resonance Sensor for Specific Detection of TNT Explosive.

PubMed

Wang, Jin; Muto, Masaki; Yatabe, Rui; Onodera, Takeshi; Tanaka, Masayoshi; Okochi, Mina; Toko, Kiyoshi

2017-09-30

In this study, a rationally-designed 2,4,6-trinitrotoluene (TNT) binding peptide derived from an amino acid sequence of the complementarity-determining region (CDR) of an anti-TNT monoclonal antibody was used for TNT detection based on a maleimide-functionalized surface plasmon resonance (SPR) sensor. By antigen-docking simulation and screening, the TNT binding candidate peptides were obtained as TNTHCDR1 derived from the heavy chain of CDR1, TNTHCDR2 derived from CDR2, and TNTHCDR3 from CDR3 of an anti-TNT antibody. The binding events between candidate peptides and TNT were evaluated using the SPR sensor by direct determination based on the 3-aminopropyltriethoxysilane (APTES) surface. The TNT binding peptide was directly immobilized on the maleimide-functionalized sensor chip surface from N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS). The results demonstrated that peptide TNTHCDR3 was identified and selected as a TNT binding peptide among the other two candidate peptides. Five kinds of TNT analogues were also investigated to testify the selectivity of TNT binding peptide TNTHCDR3. Furthermore, the results indicated that the APTES-GMBS-based SPR sensor chip procedure featured a great potential application for the direct detection of TNT.

Rational Design of Peptide-Functionalized Surface Plasmon Resonance Sensor for Specific Detection of TNT Explosive

PubMed Central

Wang, Jin; Muto, Masaki; Yatabe, Rui; Onodera, Takeshi; Okochi, Mina; Toko, Kiyoshi

2017-01-01

In this study, a rationally-designed 2,4,6-trinitrotoluene (TNT) binding peptide derived from an amino acid sequence of the complementarity-determining region (CDR) of an anti-TNT monoclonal antibody was used for TNT detection based on a maleimide-functionalized surface plasmon resonance (SPR) sensor. By antigen-docking simulation and screening, the TNT binding candidate peptides were obtained as TNTHCDR1 derived from the heavy chain of CDR1, TNTHCDR2 derived from CDR2, and TNTHCDR3 from CDR3 of an anti-TNT antibody. The binding events between candidate peptides and TNT were evaluated using the SPR sensor by direct determination based on the 3-aminopropyltriethoxysilane (APTES) surface. The TNT binding peptide was directly immobilized on the maleimide-functionalized sensor chip surface from N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS). The results demonstrated that peptide TNTHCDR3 was identified and selected as a TNT binding peptide among the other two candidate peptides. Five kinds of TNT analogues were also investigated to testify the selectivity of TNT binding peptide TNTHCDR3. Furthermore, the results indicated that the APTES-GMBS-based SPR sensor chip procedure featured a great potential application for the direct detection of TNT. PMID:28973962

Modification of Titanium Substrates with Chimeric Peptides Comprising Antimicrobial and Titanium-Binding Motifs Connected by Linkers To Inhibit Biofilm Formation.

PubMed

Liu, Zihao; Ma, Shiqing; Duan, Shun; Xuliang, Deng; Sun, Yingchun; Zhang, Xi; Xu, Xinhua; Guan, Binbin; Wang, Chao; Hu, Meilin; Qi, Xingying; Zhang, Xu; Gao, Ping

2016-03-02

Bacterial adhesion and biofilm formation are the primary causes of implant-associated infection, which is difficult to eliminate and may induce failure in dental implants. Chimeric peptides with both binding and antimicrobial motifs may provide a promising alternative to inhibit biofilm formation on titanium surfaces. In this study, chimeric peptides were designed by connecting an antimicrobial motif (JH8194: KRLFRRWQWRMKKY) with a binding motif (minTBP-1: RKLPDA) directly or via flexible/rigid linkers to modify Ti surfaces. We evaluated the binding behavior of peptides using quartz crystal microbalance (QCM) and atomic force microscopy (AFM) techniques and investigated the effect of the modification of titanium surfaces with these peptides on the bioactivity of Streptococcus gordonii (S. gordonii) and Streptococcus sanguis (S. sanguis). Compared with the flexible linker (GGGGS), the rigid linker (PAPAP) significantly increased the adsorption of the chimeric peptide on titanium surfaces (p < 0.05). Concentration-dependent adsorption is consistent with a single Langmuir model, whereas time-dependent adsorption is in line with a two-domain Langmuir model. Additionally, the chimeric peptide with the rigid linker exhibited more effective antimicrobial ability than the peptide with the flexible linker. This finding was ascribed to the ability of the rigid linker to separate functional domains and reduce their interference to the maximum extent. Consequently, the performance of chimeric peptides with specific titanium-binding motifs and antimicrobial motifs against bacteria can be optimized by the proper selection of linkers. This rational design of chimeric peptides provides a promising alternative to inhibit the formation of biofilms on titanium surfaces with the potential to prevent peri-implantitis and peri-implant mucositis.

[Preparation of anti-hCG single domain antibody by antibody grafting technique using an antigen-binding peptide].

PubMed

Peng, Jing; Wang, Qiong; Cheng, Xiaoling; Liu, Mengwen; Wang, Mei; Xin, Huawei

2018-04-25

We used the antibody grafting technology to prepare anti-hCG single-domain antibodies on the basis of antigen-binding peptide to simplify the single-domain antibody preparation process and improving the biochemical stability of peptide. By using a universal single-domain antibody backbone (cAbBCII10), CDR1 or CDR3 was replaced by the hCG-binding peptide, and the grafted antibody gene sequences were synthesized and cloned into the prokaryotic expression vector pET30a(+) in fusion with a C-terminal sfGFP gene, i.e. pET30a-(His6)-cAbBCII10-CDR1/hCGBP1-sfGFP and pET30a-(His6)-cAbBCII10-CDR3/hCGBP3-sfGFP. The recombinant plasmids were transformed into E. coli BL21(DE3), and the fusion proteins were induced by IPTG. Highly soluble recombinant fusion proteins were obtained and purified by Ni-NTA affinity column. SDS-PAGE confirmed the purified protein as the target protein. The antigen-antibody binding assay showed that both the CDR1 and CDR3 grafted antibodies have hCG-binding activities. While the titers of the two grafted antibodies were similar, the binding affinity of CDR3 grafted antibody was higher than that of CDR1 grafted protein (about 2-3 times). The grafted antibodies retained the relatively high biochemical stability of the single-domain antibody backbone and were relatively thermostable and alkaline tolerant. The obtained antibodies also had a relatively high antigen-binding specificity to hCG. This study provided a reliable experimental basis for further optimization of anti-hCG single domain antibody by antibody grafting technology using antigen-binding peptide.

Recognition of prostate-specific antigenic peptide determinants by human CD4 and CD8 T cells.

PubMed

Corman, J M; Sercarz, E E; Nanda, N K

1998-11-01

It is now becoming accepted that one is not tolerant to all the determinants of self proteins: the T cell repertoire directed to some sequences in self proteins is intact and can be activated. When a self protein is exclusively expressed by tumour cells, the T cell repertoire directed to the particular self antigen can potentially be activated to attack the tumour: this would amount to induction of a beneficial autoimmune response. Prostate cancer offers a unique opportunity for activation of a tumour-specific immune response owing to the exclusive synthesis of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) by prostatic tissue and prostate tumour cells. In this study we examine the CD4 and CD8 T cell repertoires specific for peptides of PSA and PSM in normal human male individuals, using short-term, peptide antigen-driven CD4 and CD8 T cell lines. We show that short-term, CD4 T cell lines derived from six HLA-DR4 individuals showed strong proliferative responses to six of 10 tested peptides of PSA, selected as to contain a DR4 binding motif. Short-term, CD8 T cell lines from three HLA-A1 individuals showed specific cytolytic activity for autologous targets loaded with five of five tested peptides of PSA and PSM, selected to possess an HLA-A1 binding motif. One of the peptides chosen is termed a 'dual-motif' peptide, as it encodes determinants for both CD4 and CD8 T cells. These results, indicating the existence of CD4 and CD8 T cells against determinants of the self proteins, PSA and PSM, in healthy male individuals reveal the potential of the T cell repertoire from the typical prostate cancer patient to eradicate prostate tumours upon being appropriately activated.

Food proteins and maturation of small intestinal microvillus membranes (MVM). II. Binding of gliadin hydrolysate fractions and of the gliadin peptide B3142.

PubMed

Stern, M; Gellermann, B; Belitz, H D; Wieser, H

1988-01-01

To investigate in vitro interactions between gliadin peptide fractions that have been shown to be toxic to celiac small intestinal mucosa in humans and small intestinal microvillus membranes (MVM) from rats during postnatal maturation, MVM were prepared from newborn, 18-day-old preweanling, and adult rats. Partially hydrolyzed gliadin peptide fractions B1-B4, and the pure gliadin peptide B3142 were radioiodinated and used for binding assays. Miniature ultracentrifugation was used for separation of unbound material. Binding of gliadin fractions to MVM was weak and nonspecific in terms of lacking saturation and inhibition. There was no inhibition of binding by mannan. Enzyme pretreatment of MVM (trypsin, neuraminidase, phospholipase C) did not result in any significant change of binding. Compared with peptides prepared from bovine serum albumin as a control, there was no significant difference in binding of gliadin peptide fractions to MVM. Thus, a lectin-like effect of gliadin peptides toward MVM, or the existence of a specific intestinal surface receptor for gliadin peptides appeared improbable. There were, however, consistent maturational changes in MVM binding in that newborn MVM bound more B1-B4 and B3142 compared with adult controls (p less than 0.001). Nonspecific binding of gliadin fractions to MVM might be related to the initiation of nonspecific in vitro effects of gliadin, particularly toward the immature small intestine. The MVM binding model in the rat clearly does not provide a system for studying celiac disease pathogenesis, but it might help clarify basic processes in the interaction between food-derived substances and elements of the gastrointestinal mucosal barrier.

NetMHCpan-3.0; improved prediction of binding to MHC class I molecules integrating information from multiple receptor and peptide length datasets.

PubMed

Nielsen, Morten; Andreatta, Massimo

2016-03-30

Binding of peptides to MHC class I molecules (MHC-I) is essential for antigen presentation to cytotoxic T-cells. Here, we demonstrate how a simple alignment step allowing insertions and deletions in a pan-specific MHC-I binding machine-learning model enables combining information across both multiple MHC molecules and peptide lengths. This pan-allele/pan-length algorithm significantly outperforms state-of-the-art methods, and captures differences in the length profile of binders to different MHC molecules leading to increased accuracy for ligand identification. Using this model, we demonstrate that percentile ranks in contrast to affinity-based thresholds are optimal for ligand identification due to uniform sampling of the MHC space. We have developed a neural network-based machine-learning algorithm leveraging information across multiple receptor specificities and ligand length scales, and demonstrated how this approach significantly improves the accuracy for prediction of peptide binding and identification of MHC ligands. The method is available at www.cbs.dtu.dk/services/NetMHCpan-3.0 .

Enhanced Detection of Antigen-Specific CD4+ T Cells Using Altered Peptide Flanking Residue Peptide–MHC Class II Multimers

PubMed Central

Holland, Christopher J.; Dolton, Garry; Scurr, Martin; Ladell, Kristin; Schauenburg, Andrea J.; Miners, Kelly; Madura, Florian; Sewell, Andrew K.; Price, David A.

2015-01-01

Fluorochrome-conjugated peptide–MHC (pMHC) class I multimers are staple components of the immunologist’s toolbox, enabling reliable quantification and analysis of Ag-specific CD8+ T cells irrespective of functional outputs. In contrast, widespread use of the equivalent pMHC class II (pMHC-II) reagents has been hindered by intrinsically weaker TCR affinities for pMHC-II, a lack of cooperative binding between the TCR and CD4 coreceptor, and a low frequency of Ag-specific CD4+ T cell populations in the peripheral blood. In this study, we show that peptide flanking regions, extending beyond the central nonamer core of MHC-II–bound peptides, can enhance TCR–pMHC-II binding and T cell activation without loss of specificity. Consistent with these findings, pMHC-II multimers incorporating peptide flanking residue modifications proved superior for the ex vivo detection, characterization, and manipulation of Ag-specific CD4+ T cells, highlighting an unappreciated feature of TCR–pMHC-II interactions. PMID:26553072

(99m)Tc-labeled SWL specific peptide for targeting EphA2 receptor.

PubMed

Liu, Yu; Lan, Xiaoli; Wu, Tao; Lang, Juntao; Jin, Xueyan; Sun, Xun; Wen, Qiong; An, Rui

2014-07-01

EphA2, one member of the Eph receptor family, is widely expressed in multiple aggressive cancers. SWL, a small peptide identified by phage display, has high binding affinity to EphA2, suggesting that it could be exploited for targeted molecular imaging. Therefore, a novel peptide-based probe, (99m)Tc-HYNIC-SWL, was developed and its potential to specifically target EphA2-positive tumors was investigated. The SWL peptide was labeled with hydrazinonicotinic acid (HYNIC), followed by (99m)Tc labeling. Immunofluorescence staining was carried out to detect the expression of EphA2 in A549 lung cancer cells and OCM-1 melanoma cells. Saturation binding experiments were performed by incubating A549 cells with increasing concentrations of radiolabeled peptide in vitro. To test the probe in vivo, nude mice bearing either A549 or OCM-1 derived tumors were established, injected with (99m)Tc-HYNIC-SWL, and subjected to SPECT imaging. Mice injected with excess unlabeled SWL were used as a specific control. Ex vivo γ-counting of dissected tissues from the mice was also performed to evaluate biodistribution. Immunofluorescence staining showed that A549 cells intensively expressed EphA2, while OCM-1 cells had little expression. (99m)Tc-HYNIC-SWL displayed high binding affinity with A549 cells (KD=2.6±0.7nM). From the SPECT images and the results of the biodistribution study, significantly higher uptake of the tracer was seen in A549 tumors (1.44±0.12 %ID/g) than in OCM-1 tumors (0.43±0.20 %ID/g) at 1h after injection. Pre-injection with excess unlabeled peptide in A549-bearing nude mice, significantly reduced tumor uptake of the radiolabeled probe (0.58±0.20 %ID/g) was seen. These data suggest that (99m)Tc-HYNIC-SWL specifically targets EphA2 in tumors. The expression of EphA2 can be noninvasively investigated using (99m)Tc-HYNIC-SWL by SPECT imaging. The in vitro and in vivo characteristics of (99m)Tc-HYNIC-SWL make it a promising probe for EphA2-positive tumor imaging

Use of eluted peptide sequence data to identify the binding characteristics of peptides to the insulin-dependent diabetes susceptibility allele HLA-DQ8 (DQ 3.2).

PubMed

Godkin, A; Friede, T; Davenport, M; Stevanovic, S; Willis, A; Jewell, D; Hill, A; Rammensee, H G

1997-06-01

HLA-DQ8 (A1*0301, B1*0302) and -DQ2 (A1*0501, B1*0201) are both associated with diseases such as insulin-dependent diabetes mellitus and coeliac disease. We used the technique of pool sequencing to look at the requirements of peptides binding to HLA-DQ8, and combined these data with naturally sequenced ligands and in vitro binding assays to describe a novel motif for HLA-DQ8. The motif, which has the same basic format as many HLA-DR molecules, consists of four or five anchor regions, in the positions from the N-terminus of the binding core of n, n + 3, n + 5/6 and n + 8, i.e. P1, P4, P6/7 and P9. P1 and P9 require negative or polar residues, with mainly aliphatic residues at P4 and P6/7. The features of the HLA-DQ8 motif were then compared to a pool sequence of peptides eluted from HLA-DQ2. A consensus motif for the binding of a common peptide which may be involved in disease pathogenesis is described. Neither of the disease-associated alleles HLA-DQ2 and -DQ8 have Asp at position 57 of the beta-chain. This Asp, if present, may form a salt bridge with an Arg at position 79 of the alpha-chain and so alter the binding specificity of P9. HLA-DQ2 and -DQ8 both appear to prefer negatively charged amino acids at P9. In contrast, HLA-DQ7 (A1*0301, B1*0301), which is not associated with diabetes, has Asp at beta 57, allowing positively charged amino acids at P9. This analysis of the sequence features of DQ-binding peptides suggests molecular characteristics which may be useful to predict epitopes involved in disease pathogenesis.

Toxoplasma gondii peptide ligands open the gate of the HLA class I binding groove

PubMed Central

McMurtrey, Curtis; Trolle, Thomas; Sansom, Tiffany; Remesh, Soumya G; Kaever, Thomas; Bardet, Wilfried; Jackson, Kenneth; McLeod, Rima; Sette, Alessandro; Nielsen, Morten; Zajonc, Dirk M; Blader, Ira J; Peters, Bjoern; Hildebrand, William

2016-01-01

HLA class I presentation of pathogen-derived peptide ligands is essential for CD8+ T-cell recognition of Toxoplasma gondii infected cells. Currently, little data exist pertaining to peptides that are presented after T. gondii infection. Herein we purify HLA-A*02:01 complexes from T. gondii infected cells and characterize the peptide ligands using LCMS. We identify 195 T. gondii encoded ligands originating from both secreted and cytoplasmic proteins. Surprisingly, T. gondii ligands are significantly longer than uninfected host ligands, and these longer pathogen-derived peptides maintain a canonical N-terminal binding core yet exhibit a C-terminal extension of 1–30 amino acids. Structural analysis demonstrates that binding of extended peptides opens the HLA class I F’ pocket, allowing the C-terminal extension to protrude through one end of the binding groove. In summary, we demonstrate that unrealized structural flexibility makes MHC class I receptive to parasite-derived ligands that exhibit unique C-terminal peptide extensions. DOI: http://dx.doi.org/10.7554/eLife.12556.001 PMID:26824387

Rapid Generation of a Nanocrystal-Labeled Peptide Library for Specific Identification of the Bacterium Clostrium Botulinum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tok, J B

2004-11-11

Several peptide libraries containing up to 2 million unique peptide ligands have been synthesized. The peptides are attached onto a 80 micron resin and the length of these peptide ligands ranges from 5 to 9 amino acid residues. Using a novel calorimetric assay, the libraries were screened for binding to the ganglioside-binding domain of Clostridium Tetanus Toxin, a structural similar analog of the Clostridium Botulinum toxin. Several binding peptide sequences were identified, in which the detailed binding kinetics are currently underway using the Surface Plasmon Resonance (SPR) technique.

Autoantibodies in Serum of Systemic Scleroderma Patients: Peptide-Based Epitope Mapping Indicates Increased Binding to Cytoplasmic Domains of CXCR3.

PubMed

Recke, Andreas; Regensburger, Ann-Katrin; Weigold, Florian; Müller, Antje; Heidecke, Harald; Marschner, Gabriele; Hammers, Christoph M; Ludwig, Ralf J; Riemekasten, Gabriela

2018-01-01

Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera ( N  = 32, with positive reactivity with CXCR3) to healthy controls ( N  = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients ( N  = 31) from normal healthy controls ( N  = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc

Autoantibodies in Serum of Systemic Scleroderma Patients: Peptide-Based Epitope Mapping Indicates Increased Binding to Cytoplasmic Domains of CXCR3

PubMed Central

Recke, Andreas; Regensburger, Ann-Katrin; Weigold, Florian; Müller, Antje; Heidecke, Harald; Marschner, Gabriele; Hammers, Christoph M.; Ludwig, Ralf J.; Riemekasten, Gabriela

2018-01-01

Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera (N = 32, with positive reactivity with CXCR3) to healthy controls (N = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients (N = 31) from normal healthy controls (N = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera

Attractors in Sequence Space: Agent-Based Exploration of MHC I Binding Peptides.

PubMed

Jäger, Natalie; Wisniewska, Joanna M; Hiss, Jan A; Freier, Anja; Losch, Florian O; Walden, Peter; Wrede, Paul; Schneider, Gisbert

2010-01-12

Ant Colony Optimization (ACO) is a meta-heuristic that utilizes a computational analogue of ant trail pheromones to solve combinatorial optimization problems. The size of the ant colony and the representation of the ants' pheromone trails is unique referring to the given optimization problem. In the present study, we employed ACO to generate novel peptides that stabilize MHC I protein on the plasma membrane of a murine lymphoma cell line. A jury of feedforward neural network classifiers served as fitness function for peptide design by ACO. Bioactive murine MHC I H-2K(b) stabilizing as well as nonstabilizing octapeptides were designed, synthesized and tested. These peptides reveal residue motifs that are relevant for MHC I receptor binding. We demonstrate how the performance of the implemented ACO algorithm depends on the colony size and the size of the search space. The actual peptide design process by ACO constitutes a search path in sequence space that can be visualized as trajectories on a self-organizing map (SOM). By projecting the sequence space on a SOM we visualize the convergence of the different solutions that emerge during the optimization process in sequence space. The SOM representation reveals attractors in sequence space for MHC I binding peptides. The combination of ACO and SOM enables systematic peptide optimization. This technique allows for the rational design of various types of bioactive peptides with minimal experimental effort. Here, we demonstrate its successful application to the design of MHC-I binding and nonbinding peptides which exhibit substantial bioactivity in a cell-based assay. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of glucagon-like peptide-1 receptor-binding determinants.

PubMed

Xiao, Q; Jeng, W; Wheeler, M B

2000-12-01

Glucagon-like peptide 1 (GLP-1) is a potent insulinotropic hormone currently under study as a therapeutic agent for type 2 diabetes. Since an understanding of the molecular mechanisms leading to high-affinity receptor (R) binding and activation may facilitate the development of more potent GLP-1R agonists, we have localized specific regions of GLP-1R required for binding. The purified N-terminal fragment (hereafter referred to as NT) of the GLP-1R produced in either insect (Sf9) or mammalian (COS-7) cells was shown to bind GLP-1. The physical interaction of NT with GLP-1 was first demonstrated by cross-linking ((125)I-GLP-1/NT complex band at approximately 28 kDa) and secondly by attachment to Ni(2+)-NTA beads. The GLP-1R NT protein attached to beads bound GLP-1, but with lower affinity (inhibitory concentration (IC(50)): 4.5 x 10(-7) M) than wild-type (WT) GLP-1R (IC(50): 5.2 x 10(-9)M). The low affinity of GLP-1R NT suggested that other receptor domains may contribute to GLP-1 binding. This was supported by studies using chimeric glucose-dependent insulinotropic polypeptide (GIP)/GLP-1 receptors. GIP(1-151)/GLP-1R, but not GIP(1-222)/GLP-1R, exhibited specific GLP-1 binding and GLP-1-induced cAMP production, suggesting that the region encompassing transmembrane (TM) domain 1 through to TM3 was required for binding. Since it was hypothesized that certain charged or polar amino acids in this region might be involved in binding, these residues (TM2-TM3) were analyzed by substitution mutagenesis. Five mutants (K197A, D198A, K202A, D215A, R227A) displayed remarkably reduced binding affinity. These studies indicate that the NT domain of the GLP-1R is able to bind GLP-1, but charged residues concentrated at the distal TM2/extracellular loop-1 (EC1) interface (K197, D198, K202) and in EC1 (D215 and R227) probably contribute to the binding determinants of the GLP-1R.

Chronic Beryllium Disease: Revealing the Role of Beryllium Ion and Small Peptides Binding to HLA-DP2

PubMed Central

Petukh, Marharyta; Wu, Bohua; Stefl, Shannon; Smith, Nick; Hyde-Volpe, David; Wang, Li; Alexov, Emil

2014-01-01

Chronic Beryllium (Be) Disease (CBD) is a granulomatous disorder that predominantly affects the lung. The CBD is caused by Be exposure of individuals carrying the HLA-DP2 protein of the major histocompatibility complex class II (MHCII). While the involvement of Be in the development of CBD is obvious and the binding site and the sequence of Be and peptide binding were recently experimentally revealed [1], the interplay between induced conformational changes and the changes of the peptide binding affinity in presence of Be were not investigated. Here we carry out in silico modeling and predict the Be binding to be within the acidic pocket (Glu26, Glu68 and Glu69) present on the HLA-DP2 protein in accordance with the experimental work [1]. In addition, the modeling indicates that the Be ion binds to the HLA-DP2 before the corresponding peptide is able to bind to it. Further analysis of the MD generated trajectories reveals that in the presence of the Be ion in the binding pocket of HLA-DP2, all the different types of peptides induce very similar conformational changes, but their binding affinities are quite different. Since these conformational changes are distinctly different from the changes caused by peptides normally found in the cell in the absence of Be, it can be speculated that CBD can be caused by any peptide in presence of Be ion. However, the affinities of peptides for Be loaded HLA-DP2 were found to depend of their amino acid composition and the peptides carrying acidic group at positions 4 and 7 are among the strongest binders. Thus, it is proposed that CBD is caused by the exposure of Be of an individual carrying the HLA-DP2*0201 allele and that the binding of Be to HLA-DP2 protein alters the conformational and ionization properties of HLA-DP2 such that the binding of a peptide triggers a wrong signaling cascade. PMID:25369028

The adsorption of preferential binding peptides to apatite-based materials

PubMed Central

Segvich, Sharon J.; Smith, Hayes C.; Kohn, David H.

2009-01-01

The objective of this work was to identify peptide sequences with high affinity to bone-like mineral (BLM) to provide alternative design methods for functional bone regeneration peptides. Adsorption of preferential binding peptide sequences on four apatite-based substrates [BLM and three sintered apatite disks pressed from powders containing 0% CO32− (HA), 5.6% CO32− (CA5), 10.5% CO32− (CA10)] with varied compositions and morphologies was investigated. A combination of phage display, ELISA, and computational modeling was used to elucidate three 12-mer peptide sequences APWHLSSQYSRT (A), STLPI-PHEFSRE (S), and VTKHLNQISQSY (V), from 243 candidates with preferential adsorption on BLM and HA. Overall, peptides S and V have a significantly higher adsorption to the apatite-based materials in comparison to peptide A (for S vs. A, BLM p = 0.001, CA5 p < 0.001, CA10 p < 0.001, HA p = 0.038; for V vs. A, BLM p = 0.006, CA5 p = 0.033, CA10 p = 0.029). FT-IR analysis displayed carbonate levels in CA5 and CA10 dropped to approximately 1.1–2.2% after sintering, whereas SEM imaging displayed CA5 and CA10 possess distinct morphologies. Adsorption results normalized to surface area indicate that small changes in carbonate percentage at a similar morphological scale did not provide enough carbonate incorporation to show statistical differences in peptide adsorption. Because the identified peptides (S and V) have preferential binding to apatite, their use can now be investigated in bone and dentin tissue engineering, tendon and ligament repair, and enamel formation. PMID:19095299

ERp57 interacts with conserved cysteine residues in the MHC class I peptide-binding groove.

PubMed

Antoniou, Antony N; Santos, Susana G; Campbell, Elaine C; Lynch, Sarah; Arosa, Fernando A; Powis, Simon J

2007-05-15

The oxidoreductase ERp57 is a component of the major histocompatibility complex (MHC) class I peptide-loading complex. ERp57 can interact directly with MHC class I molecules, however, little is known about which of the cysteine residues within the MHC class I molecule are relevant to this interaction. MHC class I molecules possess conserved disulfide bonds between cysteines 101-164, and 203-259 in the peptide-binding and alpha3 domain, respectively. By studying a series of mutants of these conserved residues, we demonstrate that ERp57 predominantly associates with cysteine residues in the peptide-binding domain, thus indicating ERp57 has direct access to the peptide-binding groove of MHC class I molecules during assembly.

Relationship between helix stability and binding affinities: molecular dynamics simulations of Bfl-1/A1-binding pro-apoptotic BH3 peptide helices in explicit solvent.

PubMed

Modi, Vivek; Lama, Dilraj; Sankararamakrishnan, Ramasubbu

2013-01-01

one of the interacting residues resides within the helical region. Bmf BH3 peptide with a weaker binding affinity for Bmf-1/A1 completely lost its helical character at the end of 100 ns production run and a further 50 ns simulation showed that the Bmf peptide continues to remain in random conformation. The present study clearly establishes a link between a BH3 peptide's ability to form a stable helical segment and its high binding affinity for an anti-apoptotic protein. To further test this hypothesis, we simulated a mutant Bmf peptide for 100 ns in which two residues R129 and H146 were substituted by Asn in silico in the wild-type peptide. Introduction of N-terminal Asn clearly enabled the formation of capping interactions at the N-terminus and resulted in a stable N-terminal helical segment. This demonstrates that the knowledge of interactions that help to maintain stable helical segments in a high-affinity BH3 peptide will help in designing highly specific peptide-based drugs/inhibitors. Such molecules will have the ability to bind a particular anti-apoptotic protein with high affinity.

Inhibition of Herpes Simplex Virus gD and Lymphotoxin-α Binding to HveA by Peptide Antagonists

PubMed Central

Sarrias, Maria Rosa; Whitbeck, J. Charles; Rooney, Isabelle; Spruce, Lynn; Kay, Brian K.; Montgomery, Rebecca I.; Spear, Patricia G.; Ware, Carl F.; Eisenberg, Roselyn J.; Cohen, Gary H.; Lambris, John D.

1999-01-01

The herpesvirus entry mediator A (HveA) is a recently characterized member of the tumor necrosis factor receptor family that mediates the entry of most herpes simplex virus type 1 (HSV-1) strains into mammalian cells. Studies on the interaction of HSV-1 with HveA have shown that of all the viral proteins involved in uptake, only gD has been shown to bind directly to HveA, and this binding mediates viral entry into cells. In addition to gD binding to HveA, the latter has been shown to interact with proteins of tumor necrosis factor receptor-associated factor family, lymphotoxin-α (LT-α), and a membrane-associated protein referred to as LIGHT. To study the relationship between HveA, its natural ligands, and the viral proteins involved in HSV entry into cells, we have screened two phage-displayed combinatorial peptide libraries for peptide ligands of a recombinant form of HveA. Affinity selection experiments yielded two peptide ligands, BP-1 and BP-2, which could block the interaction between gD and HveA. Of the two peptides, only BP-2 inhibited HSV entry into CHO cells transfected with an HveA-expressing plasmid. When we analyzed these peptides for the ability to interfere with HveA binding to its natural ligand LT-α, we found that BP-1 inhibited the interaction of cellular LT-α with HveA. Thus, we have dissected the sites of interaction between the cell receptor, its natural ligand LT-α and gD, the virus-specific protein involved in HSV entry into cells. PMID:10364318

Identification of Four-Jointed Box 1 (FJX1)-Specific Peptides for Immunotherapy of Nasopharyngeal Carcinoma

PubMed Central

Chai, San Jiun; Yap, Yoke Yeow; Foo, Yoke Ching; Yap, Lee Fah; Ponniah, Sathibalan; Teo, Soo Hwang; Cheong, Sok Ching; Patel, Vyomesh; Lim, Kue Peng

2015-01-01

Nasopharyngeal carcinoma (NPC) is highly prevalent in South East Asia and China. The poor outcome is due to late presentation, recurrence, distant metastasis and limited therapeutic options. For improved treatment outcome, immunotherapeutic approaches focusing on dendritic and autologous cytotoxic T-cell based therapies have been developed, but cost and infrastructure remain barriers for implementing these in low-resource settings. As our prior observations had found that four-jointed box 1 (FJX1), a tumor antigen, is overexpressed in NPCs, we investigated if short 9–20 amino acid sequence specific peptides matching to FJX1 requiring only intramuscular immunization to train host immune systems would be a better treatment option for this disease. Thus, we designed 8 FJX1-specific peptides and implemented an assay system to first, assess the binding of these peptides to HLA-A2 molecules on T2 cells. After, ELISPOT assays were used to determine the peptides immunogenicity and ability to induce potential cytotoxicity activity towards cancer cells. Also, T-cell proliferation assay was used to evaluate the potential of MHC class II peptides to stimulate the expansion of isolated T-cells. Our results demonstrate that these peptides are immunogenic and peptide stimulated T-cells were able to induce peptide-specific cytolytic activity specifically against FJX1-expressing cancer cells. In addition, we demonstrated that the MHC class II peptides were capable of inducing T-cell proliferation. Our results suggest that these peptides are capable of inducing specific cytotoxic cytokines secretion against FJX1-expressing cancer cells and serve as a potential vaccine-based therapy for NPC patients. PMID:26536470

Inhibition of gentamicin binding to rat renal brush-border membrane by megalin ligands and basic peptides.

PubMed

Nagai, Junya; Saito, Masaki; Adachi, Yoshinori; Yumoto, Ryoko; Takano, Mikihisa

2006-05-01

Our previous studies showed that coadministration of cytochrome c and a 20-residue basic peptide, N-WASP181-200 (NISHTKEKKKGKAKKKRLTK, pI=10.87) inhibits renal accumulation of gentamicin. In this study, we examined effects of ligands of megalin, an endocytic receptor involved in renal uptake of gentamicin, and basic peptides including N-WASP180-200 and its mutant peptides on gentamicin binding to isolated rat renal brush-border membrane (BBM). Gentamicin binding to BBM was inhibited by megalin ligands, basic peptide fragments of cytochrome c, and N-WASP181-200 in a concentration-dependent manner. Klotz plot analysis showed that N-WASP181-200 inhibited the binding of gentamicin in a competitive manner. By substituting glycines for lysines in N-WASP181-200 at positions 9 and 15, the inhibitory effect on gentamicin binding to BBM was reduced, which may be related to a decrease in the alpha-helix content in the peptide. Gentamicin binding to BBM treated with trypsin, in which megalin completely disappeared, was significantly but not completely decreased compared with the native BBM. In addition, treatment of BBM with trypsin led to a decrease in the inhibitory effect of N-WASP181-200 on gentamicin binding. These observations support that megalin ligands and basic peptides including N-WASP181-200 decrease renal accumulation of gentamicin by inhibiting its binding to BBM of proximal tubule cells, partly interacting with megalin. In addition, the alpha-helix conformation may play an important role in the inhibitory effect of N-WASP181-200 on the binding of gentamicin to BBM.

Trastuzumab-binding peptide display by Tobacco mosaic virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frolova, Olga Y.; Petrunia, Igor V.; Komarova, Tatiana V.

2010-11-10

Human epidermal growth factor receptor-2 (HER2/neu) is a target for the humanized monoclonal antibody trastuzumab. Recently, trastuzumab-binding peptides (TBP) of HER2/neu that inhibit proliferation of breast cancer cells were identified. We have now studied conditions of efficient assembly in vivo of Tobacco mosaic virus (TMV)-based particles displaying TBP on its surface. The system is based on an Agrobacterium-mediated co-delivery of binary vectors encoding TMV RNA and coat protein (CP) with TBP in its C-terminal extension into plant leaves. We show how the fusion of amino acid substituted TBP (sTBP) to CP via a flexible peptide linker can improve the manufacturabilitymore » of recombinant TMV (rTMV). We also reveal that rTMV particles with exposed sTBP retained trastuzumab-binding capacity but lost an anti-HER2/neu immunogenic scaffold function. Mouse antibodies against rTMV did not recognize HER2/neu on surface of human SK-BR-3 cells.« less

High-throughput screening in two dimensions: binding intensity and off-rate on a peptide microarray.

PubMed

Greving, Matthew P; Belcher, Paul E; Cox, Conor D; Daniel, Douglas; Diehnelt, Chris W; Woodbury, Neal W

2010-07-01

We report a high-throughput two-dimensional microarray-based screen, incorporating both target binding intensity and off-rate, which can be used to analyze thousands of compounds in a single binding assay. Relative binding intensities and time-resolved dissociation are measured for labeled tumor necrosis factor alpha (TNF-alpha) bound to a peptide microarray. The time-resolved dissociation is fitted to a one-component exponential decay model, from which relative dissociation rates are determined for all peptides with binding intensities above background. We show that most peptides with the slowest off-rates on the microarray also have the slowest off-rates when measured by surface plasmon resonance (SPR). 2010 Elsevier Inc. All rights reserved.

Phage-displayed peptides selected for binding to Campylobacter jejuni are antimicrobial.

PubMed

Bishop-Hurley, Sharon L; Rea, Philippa J; McSweeney, Christopher S

2010-10-01

In developed countries, Campylobacter jejuni is a leading cause of zoonotic bacterial gastroenteritis in humans with chicken meat implicated as a source of infection. Campylobacter jejuni colonises the lower gastrointestinal tract of poultry and during processing is spread from the gastrointestinal tract onto the surface of dressed carcasses. Controlling or eliminating C.jejuni on-farm is considered to be one of the best strategies for reducing human infection. Molecules on the cell surface of C.jejuni interact with the host to facilitate its colonisation and persistence in the gastrointestinal tract of poultry. We used a subtractive phage-display protocol to affinity select for peptides binding to the cell surface of a poultry isolate of C.jejuni with the aim of finding peptides that could be used to control this microorganism in chickens. In total, 27 phage peptides, representing 11 unique clones, were found to inhibit the growth of C.jejuni by up to 99.9% in vitro. One clone was bactericidal, reducing the viability of C.jejuni by 87% in vitro. The phage peptides were highly specific. They completely inhibited the growth of two of the four poultry isolates of C.jejuni tested with no activity detected towards other Gram-negative and Gram-positive bacteria.

Introducing folding stability into the score function for computational design of RNA-binding peptides boosts the probability of success.

PubMed

Xiao, Xingqing; Agris, Paul F; Hall, Carol K

2016-05-01

A computational strategy that integrates our peptide search algorithm with atomistic molecular dynamics simulation was used to design rational peptide drugs that recognize and bind to the anticodon stem and loop domain (ASL(Lys3)) of human tRNAUUULys3 for the purpose of interrupting HIV replication. The score function of the search algorithm was improved by adding a peptide stability term weighted by an adjustable factor λ to the peptide binding free energy. The five best peptide sequences associated with five different values of λ were determined using the search algorithm and then input in atomistic simulations to examine the stability of the peptides' folded conformations and their ability to bind to ASL(Lys3). Simulation results demonstrated that setting an intermediate value of λ achieves a good balance between optimizing the peptide's binding ability and stabilizing its folded conformation during the sequence evolution process, and hence leads to optimal binding to the target ASL(Lys3). Thus, addition of a peptide stability term significantly improves the success rate for our peptide design search. © 2016 Wiley Periodicals, Inc.

Binding of anti-apoptotic Bcl-2 with different BH3 peptides: A molecular dynamics study

NASA Astrophysics Data System (ADS)

Zhang, Dawei; Liu, Huihui; Cui, Jinglan

2018-01-01

In this work, molecular dynamics simulation and free energy calculations are utilized to study how different BH3 peptides originating from Bax, Bim, Bik and Noxa interact with Bcl-2, one of the main members of anti-apoptotic proteins. The effects of peptide length, sequence and helical content on the binding affinity are discussed, on which a novel BH3-like peptide is designed in silico with an improved binding property.

Highly potent antimicrobial peptides from N-terminal membrane-binding region of E. coli MreB.

PubMed

Saikia, Karabi; Sravani, Yalavarthi Durga; Ramakrishnan, Vibin; Chaudhary, Nitin

2017-02-23

Microbial pathogenesis is a serious health concern. The threat escalates as the existing conventional antimicrobials are losing their efficacy against the evolving pathogens. Peptides hold promise to be developed into next-generation antibiotics. Antimicrobial peptides adopt amphipathic structures that could selectively bind to and disrupt the microbial membranes. Interaction of proteins with membranes is central to all living systems and we reasoned that the membrane-binding domains in microbial proteins could be developed into efficient antimicrobials. This is an interesting approach as self-like sequences could elude the microbial strategies of degrading the antimicrobial peptides, one of the mechanisms of showing resistance to antimicrobials. We selected the 9-residue-long membrane-binding region of E. coli MreB protein. The 9-residue peptide (C-terminal amide) and its N-terminal acetylated analog displayed broad-spectrum activity, killing Gram-negative bacteria, Gram-positive bacteria, and fungi. Extension with a tryptophan residue at the N-terminus drastically improved the activity of the peptides with lethal concentrations ≤10 μM against all the organisms tested. The tryptophan-extended peptides caused complete killing of C. albicans as well as gentamicin and methicillin resistant S. aureus at 5 μM concentration. Lipid-binding studies and electron microscopic analyses of the peptide-treated microbes suggest membrane disruption as the mechanism of killing.

Bio-inorganic synthesis of ZnO powders using recombinant His-tagged ZnO binding peptide as a promoter.

PubMed

Song, Lei; Liu, Yingying; Zhang, Zhifang; Wang, Xi; Chen, Jinchun

2010-10-01

Inorganic-binding peptides termed as genetically engineered polypeptides for inorganics (GEPIs), are small peptide sequences selected via combinatorial biology-based protocols of phage or cell surface display technologies. Recent advances in nanotechnology and molecular biology allow the engineering of these peptides with specific affinity to inorganics, often used as molecular linkers or assemblers, to facilitate materials synthesis, which provides a new insight into the material science and engineering field. As a case study on this biomimetic application, here we report a novel biosynthetic ZnO binding protein and its application in promoting bio-inorganic materials synthesis. In brief, the gene encoding a ZnO binding peptide(ZBP) was genetically fused with His(6)-tag and GST-tag using E.coli expression vector pET-28a (+) and pGEX-4T-3. The recombinant protein GST-His-ZBP was expressed, purified with Ni-NTA system, identified by SDS-PAGE electrophoresis and Western blot analysis and confirmed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Affinity adsorption test demonstrated that the fusion protein had a specific avidity for ZnO nanoparticles (NPs). Results from the bio-inorganic synthesis experiment indicated that the new protein played a promoting part in grain refinement and accelerated precipitation during the formation of the ultra-fine precursor powders in the Zn(OH)(2) sol. X-ray diffraction (XRD) analysis on the final products after calcining the precursor powders showed that hexagonal wurtzite ZnO crystals were obtained. Our work suggested a novel approach to the application about the organic-inorganic interactions.

The common equine class I molecule Eqca-1*00101 (ELA-A3.1) is characterized by narrow peptide binding and T cell epitope repertoires.

PubMed

Bergmann, Tobias; Moore, Carrie; Sidney, John; Miller, Donald; Tallmadge, Rebecca; Harman, Rebecca M; Oseroff, Carla; Wriston, Amanda; Shabanowitz, Jeffrey; Hunt, Donald F; Osterrieder, Nikolaus; Peters, Bjoern; Antczak, Douglas F; Sette, Alessandro

2015-11-01

Here we describe a detailed quantitative peptide-binding motif for the common equine leukocyte antigen (ELA) class I allele Eqca-1*00101, present in roughly 25 % of Thoroughbred horses. We determined a preliminary binding motif by sequencing endogenously bound ligands. Subsequently, a positional scanning combinatorial library (PSCL) was used to further characterize binding specificity and derive a quantitative motif involving aspartic acid in position 2 and hydrophobic residues at the C-terminus. Using this motif, we selected and tested 9- and 10-mer peptides derived from the equine herpesvirus type 1 (EHV-1) proteome for their capacity to bind Eqca-1*00101. PSCL predictions were very efficient, with an receiver operating characteristic (ROC) curve performance of 0.877, and 87 peptides derived from 40 different EHV-1 proteins were identified with affinities of 500 nM or higher. Quantitative analysis revealed that Eqca-1*00101 has a narrow peptide-binding repertoire, in comparison to those of most human, non-human primate, and mouse class I alleles. Peripheral blood mononuclear cells from six EHV-1-infected, or vaccinated but uninfected, Eqca-1*00101-positive horses were used in IFN-γ enzyme-linked immunospot (ELISPOT) assays. When we screened the 87 Eqca-1*00101-binding peptides for T cell reactivity, only one Eqca-1*00101 epitope, derived from the intermediate-early protein ICP4, was identified. Thus, despite its common occurrence in several horse breeds, Eqca-1*00101 is associated with a narrow binding repertoire and a similarly narrow T cell response to an important equine viral pathogen. Intriguingly, these features are shared with other human and macaque major histocompatibility complex (MHC) molecules with a similar specificity for D in position 2 or 3 in their main anchor motif.

The common equine class I molecule Eqca-1*00101 (ELA-A3.1) is characterized by narrow peptide binding and T cell epitope repertoires

PubMed Central

Bergmann, Tobias; Moore, Carrie; Sidney, John; Miller, Donald; Tallmadge, Rebecca; Harman, Rebecca M.; Oseroff, Carla; Wriston, Amanda; Shabanowitz, Jeffrey; Hunt, Donald F.; Osterrieder, Nikolaus; Peters, Bjoern; Antczak, Douglas F.; Sette, Alessandro

2016-01-01

Here we describe a detailed quantitative peptide-binding motif for the common equine leukocyte antigen (ELA) class I allele Eqca-1*00101, present in roughly 25 % of Thoroughbred horses. We determined a preliminary binding motif by sequencing endogenously bound ligands. Subsequently, a positional scanning combinatorial library (PSCL) was used to further characterize binding specificity and derive a quantitative motif involving aspartic acid in position 2 and hydrophobic residues at the C-terminus. Using this motif, we selected and tested 9- and 10-mer peptides derived from the equine herpesvirus type 1 (EHV-1) proteome for their capacity to bind Eqca-1*00101. PSCL predictions were very efficient, with an receiver operating characteristic (ROC) curve performance of 0.877, and 87 peptides derived from 40 different EHV-1 proteins were identified with affinities of 500 nM or higher. Quantitative analysis revealed that Eqca-1*00101 has a narrow peptide-binding repertoire, in comparison to those of most human, non-human primate, and mouse class I alleles. Peripheral blood mononuclear cells from six EHV-1-infected, or vaccinated but uninfected, Eqca-1*00101-positive horses were used in IFN-γ enzyme-linked immunospot (ELISPOT) assays. When we screened the 87 Eqca-1*00101-binding peptides for T cell reactivity, only one Eqca-1*00101 epitope, derived from the intermediate-early protein ICP4, was identified. Thus, despite its common occurrence in several horse breeds, Eqca-1*00101 is associated with a narrow binding repertoire and a similarly narrow T cell response to an important equine viral pathogen. Intriguingly, these features are shared with other human and macaque major histocompatibility complex (MHC) molecules with a similar specificity for D in position 2 or 3 in their main anchor motif. PMID:26399241

Two classes of receptor specific for sperm-activating peptide III in sand-dollar spermatozoa.

PubMed

Yoshino, K; Suzuki, N

1992-06-15

We characterized receptors specific for sperm-activating peptide III (SAP-III: DSDSAQNLIQ) in spermatozoa of the sand dollar, Clypeaster japonicus, using both binding and cross-linking techniques. Analyses of the data obtained from the equilibrium binding of a radiolabeled SAP-III analogueto C. japonicus spermatozoa, using Klotz, Scatchard and Hill plots, showed the presence of two classes of receptors specific for SAP-III in the spermatozoa. One of the receptors (high-affinity) had a Kd of 3.4 nM and 3.4 x 10(4) binding sites/spermatozoon. The other receptor (low-affinity) had a Kd of 48 nM, with 6.1 x 10(4) binding sites/spermatozoon. The Kd of the high-affinity receptor was comparable to the median effective concentration of the intracellular-pH-increasing activity of SAP-III and that of the low-affinity receptor was comparable to the median effective concentration of the cellular-cGMP-elevating activity of the peptide. In addition, Scatchard and Hill plots of the data suggested the existence of positive cooperativity between the high-affinity members. Similar results were also obtained from a binding experiment using a sperm-membrane fraction prepared from C. japonicus spermatozoa. The incubation of intact spermatozoa or sperm plasma membranes with the radioiodinated SAP-III analogue and a chemical cross-linking reagent, disuccinimidyl suberate, resulted in the radiolabeling of three proteins with molecular masses of 126, 87 and 64 kDa, estimated by SDS/PAGE under reducing conditions.

Spectroscopic and biological activity studies of the chromium-binding peptide EEEEGDD.

PubMed

Arakawa, Hirohumi; Kandadi, Machender R; Panzhinskiy, Evgeniy; Belmore, Kenneth; Deng, Ge; Love, Ebony; Robertson, Preshus M; Commodore, Juliette J; Cassady, Carolyn J; Nair, Sreejayan; Vincent, John B

2016-06-01

While trivalent chromium has been shown at high doses to have pharmacological effects improving insulin resistance in rodent models of insulin resistance, the mechanism of action of chromium at a molecular level is not known. The chromium-binding and transport agent low-molecular-weight chromium-binding substance (LMWCr) has been proposed to be the biologically active form of chromium. LMWCr has recently been shown to be comprised of a heptapeptide of the sequence EEEEDGG. The binding of Cr(3+) to this heptapeptide has been examined. Mass spectrometric and a variety of spectroscopic studies have shown that multiple chromic ions bind to the peptide in an octahedral fashion through carboxylate groups and potentially small anionic ligands such as oxide and hydroxide. A complex of Cr and the peptide when administered intravenously to mice is able to decrease area under the curve in intravenous glucose tolerance tests. It can also restore insulin-stimulated glucose uptake in myotubes rendered insulin resistant by treating them with a high-glucose media.

Binding of calcium and target peptide to calmodulin-like protein CML19, the centrin 2 of Arabidopsis thaliana.

PubMed

La Verde, Valentina; Trande, Matteo; D'Onofrio, Mariapina; Dominici, Paola; Astegno, Alessandra

2018-03-01

Calmodulin-like protein 19 (CML19) is an Arabidopsis centrin that modulates nucleotide excision repair (NER) by binding to RAD4 protein, the Arabidopsis homolog of human Xeroderma pigmentosum complementation group C protein. Although the necessity of CML19 as a part of the RAD4 plant recognition complex for functional NER is known at a cellular level, little is known at a molecular level. Herein, we used a combination of biophysical and biochemical approaches to investigate the structural and ion and target-peptide binding properties of CML19. We found that CML19 possesses four Ca 2+ -specific binding sites, two of high affinity in the N-terminal domain and two of low affinity in the C-terminal domain. Binding of Ca 2+ to CML19 increases its alpha-helix content, stabilizes the tertiary structure, and triggers a conformational change, resulting in the exposure of a hydrophobic patch instrumental for target protein recognition. Using bioinformatics tools we identified a CML19-binding site at the C-terminus of RAD4, and through in vitro binding experiments we analyzed the interaction between a 17-mer peptide representing this site and CML19. We found that the peptide shows a high affinity for CML19 in the presence of Ca 2+ (stoichiometry 1:1) and the interaction primarily involves the C-terminal half of CML19. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression and GTP sensitivity of peptide histidine isoleucine high-affinity-binding sites in rat.

PubMed

Debaigt, Colin; Meunier, Annie-Claire; Goursaud, Stephanie; Montoni, Alicia; Pineau, Nicolas; Couvineau, Alain; Laburthe, Marc; Muller, Jean-Marc; Janet, Thierry

2006-07-01

High-affinity-binding sites for the vasoactive intestinal peptide (VIP) analogs peptide histidine/isoleucine-amide (PHI)/carboxyterminal methionine instead of isoleucine (PHM) are expressed in numerous tissues in the body but the nature of their receptors remains to be elucidated. The data presented indicate that PHI discriminated a high-affinity guanosine 5'-triphosphate (GTP)-insensitive-binding subtype that represented the totality of the PHI-binding sites in newborn rat tissues but was differentially expressed in adult animals. The GTP-insensitive PHI/PHM-binding sites were also observed in CHO cells over expressing the VPAC2 but not the VPAC1 VIP receptor.

A peptide sequence on carcinoembryonic antigen binds to a 80kD protein on Kupffer cells.

PubMed

Thomas, P; Petrick, A T; Toth, C A; Fox, E S; Elting, J J; Steele, G

1992-10-30

Clearance of carcinoembryonic antigen (CEA) from the circulation is by binding to Kupffer cells in the liver. We have shown that CEA binding to Kupffer cells occurs via a peptide sequence YPELPK representing amino acids 107-112 of the CEA sequence. This peptide sequence is located in the region between the N-terminal and the first immunoglobulin like loop domain. Using native CEA and peptides containing this sequence complexed with a heterobifunctional crosslinking agent and ligand blotting with biotinylated CEA and NCA we have shown binding to an 80kD protein on the Kupffer cell surface. This binding protein may be important in the development of hepatic metastases.

Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

PubMed Central

Choe, Weonu; Durgannavar, Trishaladevi A.; Chung, Sang J.

2016-01-01

The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed. PMID:28774114

Novel Interactions of the TRTK12 Peptide with S100 Protein Family Members: Specificity and Thermodynamic Characterization

PubMed Central

Wafer, Lucas N.; Tzul, Franco O.; Pandharipande, Pranav P.; Makhatadze, George I.

2013-01-01

The S100 protein family consists of small, dimeric proteins that exert their biological functions in response to changing calcium concentrations. S100B is the best studied member and has been shown to interact with over 20 binding partners in a calcium-dependent manner. The TRTK12 peptide, derived from the consensus binding sequence for S100B, has previously been found to interact with S100A1 and has been proposed to be a general binding partner of the S100 family. To test this hypothesis and gain a better understanding of the specificity of binding for the S100 proteins sixteen members of the human S100 family were screened against this peptide and its alanine variants. Novel interactions were only found with two family members: S100P and S100A2, indicating that TRTK12 selectively interacts with a small subset of the S100 proteins. Substantial promiscuity was observed in the binding site of S100B to accommodate variations in the peptide sequence, while S100A1, S100A2, and S100P exhibited larger differences in the binding constants for the TRTK12 alanine variants. This suggests that single-point substitutions can be used to selectively modulate the affinity of TRTK12 peptides for individual S100 proteins. This study has important implications for the rational drug design of inhibitors for the S100 proteins, which are involved in a variety of cancers and neurodegenerative diseases. PMID:23899389

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor*

PubMed Central

Lawrence, Callum F.; Margetts, Mai B.; Menting, John G.; Smith, Nicholas A.; Smith, Brian J.; Ward, Colin W.; Lawrence, Michael C.

2016-01-01

Sets of synthetic peptides that interact with the insulin receptor ectodomain have been discovered by phage display and reported in the literature. These peptides were grouped into three classes termed Site 1, Site 2, and Site 3 based on their mutual competition of binding to the receptor. Further refinement has yielded, in particular, a 36-residue Site 2-Site 1 fusion peptide, S519, that binds the insulin receptor with subnanomolar affinity and exhibits agonist activity in both lipogenesis and glucose uptake assays. Here, we report three-dimensional crystallographic detail of the interaction of the C-terminal, 16-residue Site 1 component (S519C16) of S519 with the first leucine-rich repeat domain (L1) of the insulin receptor. Our structure shows that S519C16 binds to the same site on the L1 surface as that occupied by a critical component of the primary binding site, namely the helical C-terminal segment of the insulin receptor α-chain (termed αCT). In particular, the two phenylalanine residues within the FYXWF motif of S519C16 are seen to engage the insulin receptor L1 domain surface in a fashion almost identical to the respective αCT residues Phe701 and Phe705. The structure provides a platform for the further development of peptidic and/or small molecule agents directed toward the insulin receptor and/or the type 1 insulin-like growth factor receptor. PMID:27281820

Knowledge-based grouping of modeled HLA peptide complexes.

PubMed

Kangueane, P; Sakharkar, M K; Lim, K S; Hao, H; Lin, K; Chee, R E; Kolatkar, P R

2000-05-01

Human leukocyte antigens are the most polymorphic of human genes and multiple sequence alignment shows that such polymorphisms are clustered in the functional peptide binding domains. Because of such polymorphism among the peptide binding residues, the prediction of peptides that bind to specific HLA molecules is very difficult. In recent years two different types of computer based prediction methods have been developed and both the methods have their own advantages and disadvantages. The nonavailability of allele specific binding data restricts the use of knowledge-based prediction methods for a wide range of HLA alleles. Alternatively, the modeling scheme appears to be a promising predictive tool for the selection of peptides that bind to specific HLA molecules. The scoring of the modeled HLA-peptide complexes is a major concern. The use of knowledge based rules (van der Waals clashes and solvent exposed hydrophobic residues) to distinguish binders from nonbinders is applied in the present study. The rules based on (1) number of observed atomic clashes between the modeled peptide and the HLA structure, and (2) number of solvent exposed hydrophobic residues on the modeled peptide effectively discriminate experimentally known binders from poor/nonbinders. Solved crystal complexes show no vdW Clash (vdWC) in 95% cases and no solvent exposed hydrophobic peptide residues (SEHPR) were seen in 86% cases. In our attempt to compare experimental binding data with the predicted scores by this scoring scheme, 77% of the peptides are correctly grouped as good binders with a sensitivity of 71%.

Delineation of the peptide binding site of the human galanin receptor.

PubMed Central

Kask, K; Berthold, M; Kahl, U; Nordvall, G; Bartfai, T

1996-01-01

Galanin, a neuroendocrine peptide of 29 amino acids, binds to Gi/Go-coupled receptors to trigger cellular responses. To determine which amino acids of the recently cloned seven-transmembrane domain-type human galanin receptor are involved in the high-affinity binding of the endogenous peptide ligand, we performed a mutagenesis study. Mutation of the His264 or His267 of transmembrane domain VI to alanine, or of Phe282 of transmembrane domain VII to glycine, results in an apparent loss of galanin binding. The substitution of Glu271 to serine in the extracellular loop III of the receptor causes a 12-fold loss in affinity for galanin. We combined the mutagenesis results with data on the pharmacophores (Trp2, Tyr9) of galanin and with molecular modelling of the receptor using bacteriorhodopsin as a model. Based on these studies, we propose a binding site model for the endogenous peptide ligand in the galanin receptor where the N-terminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+ sensitive pair of His264 and His267 of transmembrane domain VI, and Tyr9 of galanin interacts with Phe282 of transmembrane domain VII, while the C-terminus of galanin is pointing towards the N-terminus of th Images PMID:8617199

Proximity-Induced Covalent Labeling of Proteins with a Reactive Fluorophore-Binding Peptide Tag.

PubMed

Sunbul, Murat; Nacheva, Lora; Jäschke, Andres

2015-08-19

Labeling of proteins with fluorescent dyes in live cells enables the investigation of their roles in biological systems by fluorescence microscopy. Because the labeling procedure should not disturb the native function of the protein of interest, it is of high importance to find the optimum labeling method for the problem to be studied. Here, we developed a rapid one-step method to covalently and site-specifically label proteins with a TexasRed fluorophore in vitro and in live bacteria. To this end, a genetically encodable TexasRed fluorophore-binding peptide (TR512) was converted into a reactive tag (ReacTR) by adjoining a cysteine residue which rapidly reacts with N-α-chloroacetamide-conjugated TexasRed fluorophore owing to the proximity effect; ReacTR tag first binds to the TexasRed fluorophore and this interaction brings the nucleophilic cysteine and the electrophilic N-α-chloroacetamide groups in close proximity. Our method has several advantages over existing methods: (i) it utilizes a peptide tag much smaller than fluorescent proteins, the SNAP, CLIP, or HaLo tags; (ii) it allows for labeling of proteins with a small, photostable, red-emitting TexasRed fluorophore; (iii) the probe used is very easy to synthesize; (iv) no enzyme is required to transfer the fluorophore to the peptide tag; and (v) labeling yields a stable covalent product in a very fast reaction.

Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity

PubMed Central

Cole, David K.; Bulek, Anna M.; Dolton, Garry; Schauenberg, Andrea J.; Szomolay, Barbara; Trimby, Andrew; Jothikumar, Prithiviraj; Fuller, Anna; Skowera, Ania; Rossjohn, Jamie; Zhu, Cheng; Miles, John J.; Wooldridge, Linda; Rizkallah, Pierre J.; Sewell, Andrew K.

2016-01-01

The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease. PMID:27183389

Exploring the impact of the side-chain length on peptide/RNA binding events.

PubMed

Sbicca, Lola; González, Alejandro López; Gresika, Alexandra; Di Giorgio, Audrey; Closa, Jordi Teixido; Tejedor, Roger Estrada; Andréola, Marie-Line; Azoulay, Stéphane; Patino, Nadia

2017-07-19

The impact of the amino-acid side-chain length on peptide-RNA binding events has been investigated using HIV-1 Tat derived peptides as ligands and the HIV-1 TAR RNA element as an RNA model. Our studies demonstrate that increasing the length of all peptide side-chains improves unexpectedly the binding affinity (K D ) but reduces the degree of compactness of the peptide-RNA complex. Overall, the side-chain length appears to modulate in an unpredictable way the ability of the peptide to compete with the cognate TAR RNA partner. Beyond the establishment of non-intuitive fundamental relationships, our results open up new perspectives in the design of effective RNA ligand competitors, since a large number of them have already been identified but few studies report on the modulation of the biological activity by modifying in the same way the length of all chains connecting RNA recognition motives to the central scaffold of a ligand.

SPECT imaging of fibrin using fibrin-binding peptides.

PubMed

Starmans, Lucas W E; van Duijnhoven, Sander M J; Rossin, Raffaella; Aime, Silvio; Daemen, Mat J A P; Nicolay, Klaas; Grüll, Holger

2013-01-01

Noninvasive detection of fibrin in vivo using diagnostic imaging modalities may improve clinical decision-making on possible therapeutic options in atherosclerosis, cancer and thrombus-related pathologies such as pulmonary embolism and deep venous thrombosis. The aim of this study was to assess the potential of a novel (111)In-labeled fibrin-binding peptide (FibPep) to visualize thrombi in mice noninvasively using single-photon emission computed tomography (SPECT). FibPep and a negative control peptide (NCFibPep) were synthesized and their fibrin-binding properties were assessed in vitro. FibPep showed enhanced binding compared with NCFibPep to both fibrin and blood clots. FibPep bound to fibrin with a dissociation constant (K(d)) of 0.8 μ m, whereas NCFibPep displayed at least a 100-fold lower affinity towards fibrin. A FeCl3 -injury carotid artery thrombosis mouse model was used to evaluate the peptides in vivo. FibPep and NCFibPep displayed rapid blood clearance and were eliminated via the renal pathway. In vivo SPECT imaging using FibPep allowed clear visualization of thrombi. Ex vivo biodistribution showed significantly increased uptake of FibPep in the thrombus-containing carotid in comparison to the noninjured carotid (5.7 ± 0.7 and 0.6 ± 0.4% injected dose per gram (%ID g(-1)), respectively; p < 0.01; n = 4), whereas nonspecific NCFibPep did not (0.4 ± 0.2 and 0.3 ± 0.0%ID g(-1), respectively; n = 4). In conclusion, FibPep displayed high affinity towards fibrin in vitro and rapid blood clearance in vivo, and allowed sensitive detection of thrombi using SPECT imaging. Therefore, this particular imaging approach may provide a new tool to diagnose and monitor diseases such as atherosclerosis and cancer. Copyright © 2013 John Wiley & Sons, Ltd.

Conformational Contribution to Thermodynamics of Binding in Protein-Peptide Complexes through Microscopic Simulation

PubMed Central

Das, Amit; Chakrabarti, J.; Ghosh, Mahua

2013-01-01

We extract the thermodynamics of conformational changes in biomacromolecular complexes from the distributions of the dihedral angles of the macromolecules. These distributions are obtained from the equilibrium configurations generated via all-atom molecular dynamics simulations. The conformational thermodynamics data we obtained for calmodulin-peptide complexes using our methodology corroborate well with the experimentally observed conformational and binding entropies. The conformational free-energy changes and their contributions for different peptide-binding regions of calmodulin are evaluated microscopically. PMID:23528087

Enhanced In Vivo Tumor Detection by Active Tumor Cell Targeting Using Multiple Tumor Receptor-Binding Peptides Presented on Genetically Engineered Human Ferritin Nanoparticles.

PubMed

Kwon, Koo Chul; Ko, Ho Kyung; Lee, Jiyun; Lee, Eun Jung; Kim, Kwangmeyung; Lee, Jeewon

2016-08-01

Human ferritin heavy-chain nanoparticle (hFTH) is genetically engineered to present tumor receptor-binding peptides (affibody and/or RGD-derived cyclic peptides, named 4CRGD here) on its surface. The affibody and 4CRGD specifically and strongly binds to human epidermal growth factor receptor I (EGFR) and human integrin αvβ3, respectively, which are overexpressed on various tumor cells. Through in vitro culture of EGFR-overexpressing adenocarcinoma (MDA-MB-468) and integrin-overexpressing glioblastoma cells (U87MG), it is clarified that specific interactions between receptors on tumor cells and receptor-binding peptides on engineered hFTH is critical in active tumor cell targeting. After labeling with the near-infrared fluorescence dye (Cy5.5) and intravenouse injection into MDA-MB-468 or U87MG tumor-bearing mice, the recombinant hFTHs presenting either peptide or both of affibody and 4CRGD are successfully delivered to and retained in the tumor for a prolonged period of time. In particular, the recombinant hFTH presenting both affibody and 4CRGD notably enhances in vivo detection of U87MG tumors that express heterogeneous receptors, integrin and EGFR, compared to the other recombinant hFTHs presenting either affibody or 4CRGD only. Like affibody and 4CRGD used in this study, other multiple tumor receptor-binding peptides can be also genetically introduced to the hFTH surface for actively targeting of in vivo tumors with heterogenous receptors. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

NASA Astrophysics Data System (ADS)

Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

2017-07-01

The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

gamma. -Preprotachykinin-(72-92)-peptide amide: An endogenous preprotachykinin I gene-derived peptide that preferentially binds to neurokinin-2 receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dam, T.V.; Takeda, Y.; Krause, J.E.

1990-01-01

The presence of N-terminally extended forms of neurokinin A has recently been reported in the mammalian brain. Among them, gamma-preprotachykinin-(72-92)-peptide amide (gamma-PPT-(72-92)-NH2), a peptide derived by posttranslational processing of gamma-preprotachykinin, is most prominent. We report here that this peptide most likely acts on neurokinin-2 receptor sites since neurokinin A (a putative neurokinin-2 agonist) and gamma-PPT-(72-92)-NH2 are potent competitors of 125I-labeled gamma-PPT-(72-92)-NH2 binding whereas selective neurokinin-1 and -3 agonists are not. Moreover, the distribution of 125I-labeled gamma-PPT-(72-92)-NH2 and 125I-labeled neurokinin A binding sites are very similar in rat brain. On the other hand, 125I-labeled Bolton-Hunter-substance P (a neurokinin-1 ligand) and 125I-labeledmore » Bolton-Hunter-eledoisin (a neurokinin-3 ligand) binding sites are differentially located in this tissue. Thus, it appears that gamma-PPT-(72-92)-NH2 binds to neurokinin-2 receptors and should be considered as a putative endogenous ligand for this receptor class.« less

The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis.

PubMed

Mardirossian, Mario; Grzela, Renata; Giglione, Carmela; Meinnel, Thierry; Gennaro, Renato; Mergaert, Peter; Scocchi, Marco

2014-12-18

Antimicrobial peptides (AMPs) are molecules from innate immunity with high potential as novel anti-infective agents. Most of them inactivate bacteria through pore formation or membrane barrier disruption, but others cross the membrane without damages and act inside the cells, affecting vital processes. However, little is known about their intracellular bacterial targets. Here we report that Bac71-35, a proline-rich AMP belonging to the cathelicidin family, can reach high concentrations (up to 340 μM) inside the E. coli cytoplasm. The peptide specifically and completely inhibits in vitro translation in the micromolar concentration range. Experiments of incorporation of radioactive precursors in macromolecules with E. coli cells confirmed that Bac71-35 affects specifically protein synthesis. Ribosome coprecipitation and crosslinking assays showed that the peptide interacts with ribosomes, binding to a limited subset of ribosomal proteins. Overall, these results indicate that the killing mechanism of Bac71-35 is based on a specific block of protein synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Discovery of high-affinity BCL6-binding peptide and its structure-activity relationship

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakamoto, Kotaro; Sogabe, Satoshi; Kamada, Yusuke

B cell lymphoma 6 (BCL6) is a transcriptional repressor that interacts with its corepressors BcoR and SMRT. Since this protein-protein interaction (PPI) induces activation and differentiation of B lymphocytes, BCL6 has been an attractive drug target for potential autoimmune disease treatments. Here we report a novel BCL6 inhibitory peptide, F1324 (Ac-LWYTDIRMSWRVP-OH), which we discovered using phage display technology; we also discuss this peptide's structure-activity relationship (SAR). For BCL6(5-129) binding, K{sub D} and IC{sub 50} values of F1324 were 0.57 nM and 1 nM according to the results of an SPR analysis and cell-free ELISA assay, respectively. In contrast, BcoR(Arg498-514Pro) and SMRT(Leu1422-Arg1438) exhibitedmore » relatively weak micromole-order binding to BCL6. Furthermore, Fusion protein AcGFP-F1324 transiently expressed in HEK293T cells inhibited intracellular PPI in cell-based M2H assay. By examination of the truncation and fragmentation of F1324, the C-terminal sequence WRVP, which is similar to the BcoR(509-512) sequence WVVP, was identified as being critical for BCL6 binding. In addition, subsequent single-crystal X-ray diffraction analysis of F1324/BCL6(5-129) complex revealed that the high affinity of F1324 was caused by effective interaction of its side chains while its main chain structure was similar to that of BcoR(Arg498-514Pro). To our knowledge, F1324 is the strongest BCL6-binding peptide yet reported. - Highlights: • F1324 was discovered as 5000-times higher affinity peptide to BCL6 than that of BcoR(R498-P514). • X-ray crystal structure analysis revealed the binding mode. • To our knowledge, F1324 is the strongest BCL6-binding and -inhibition peptide so far.« less

ARSENITE BINDING TO SYNTHETIC PEPTIDES: THE EFFECT OF INCREASING LENGTH BETWEEN TWO CYSTEINES

EPA Science Inventory

Binding of trivalent arsenicals to peptides and proteins can alter peptide/protein structure and enzyme function and thereby contribute to arsenic toxicity and carcinogenicity. We utilized radioactive 73As- labeled arsenite and vacuum filtration methodology to determine the bindi...

The development and application of a quantitative peptide microarray platform to SH2 domain specificity space

NASA Astrophysics Data System (ADS)

Engelmann, Brett Warren

The Src homology 2 (SH2) domains evolved alongside protein tyrosine kinases (PTKs) and phosphatases (PTPs) in metazoans to recognize the phosphotyrosine (pY) post-translational modification. The human genome encodes 121 SH2 domains within 111 SH2 domain containing proteins that represent the primary mechanism for cellular signal transduction immediately downstream of PTKs. Despite pY recognition contributing to roughly half of the binding energy, SH2 domains possess substantial binding specificity, or affinity discrimination between phosphopeptide ligands. This specificity is largely imparted by amino acids (AAs) adjacent to the pY, typically from positions +1 to +4 C-terminal to the pY. Much experimental effort has been undertaken to construct preferred binding motifs for many SH2 domains. However, due to limitations in previous experimental methodologies these motifs do not account for the interplay between AAs. It was therefore not known how AAs within the context of individual peptides function to impart SH2 domain specificity. In this work we identified the critical role context plays in defining SH2 domain specificity for physiological ligands. We also constructed a high quality interactome using 50 SH2 domains and 192 physiological ligands. We next developed a quantitative high-throughput (Q-HTP) peptide microarray platform to assess the affinities four SH2 domains have for 124 physiological ligands. We demonstrated the superior characteristics of our platform relative to preceding approaches and validated our results using established biophysical techniques, literature corroboration, and predictive algorithms. The quantitative information provided by the arrays was leveraged to investigate SH2 domain binding distributions and identify points of binding overlap. Our microarray derived affinity estimates were integrated to produce quantitative interaction motifs capable of predicting interactions. Furthermore, our microarrays proved capable of resolving

Photounbinding of Calmodulin from a Family of CaM Binding Peptides

PubMed Central

Neumüller, Klaus G.; Elsayad, Kareem; Reisecker, Johannes M.; Waxham, M. Neal; Heinze, Katrin G.

2010-01-01

Background Recent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light. The mechanism responsible for the photounbinding effect, however, remains elusive. Here, we give important insights into the mechanism of photounbinding and show that the effect is not restricted to antibody/antigen binding. Methodology/Principal Findings We present studies of the photounbinding of labeled calmodulin (CaM) from a set of CaM-binding peptides with different affinities to CaM after one- and two-photon excitation. We found that the photounbinding effect becomes stronger with increasing binding affinity. Our observation that photounbinding can be influenced by using free radical scavengers, that it does not occur with either unlabeled protein or non-fluorescent quencher dyes, and that it becomes evident shortly after or with photobleaching suggest that photounbinding and photobleaching are closely linked. Conclusions/Significance The experimental results exclude surface effects, or heating by laser irradiation as potential causes of photounbinding. Our data suggest that free radicals formed through photobleaching may cause a conformational change of the CaM which lowers their binding affinity with the peptide or its respective binding partner. PMID:21124984

Dimensionality of Carbon Nanomaterials Determines the Binding and Dynamics of Amyloidogenic Peptides: Multiscale Theoretical Simulations

PubMed Central

Hine, Nicholas D. M.; Mostofi, Arash A.; Yarovsky, Irene

2013-01-01

Experimental studies have demonstrated that nanoparticles can affect the rate of protein self-assembly, possibly interfering with the development of protein misfolding diseases such as Alzheimer's, Parkinson's and prion disease caused by aggregation and fibril formation of amyloid-prone proteins. We employ classical molecular dynamics simulations and large-scale density functional theory calculations to investigate the effects of nanomaterials on the structure, dynamics and binding of an amyloidogenic peptide apoC-II(60-70). We show that the binding affinity of this peptide to carbonaceous nanomaterials such as C60, nanotubes and graphene decreases with increasing nanoparticle curvature. Strong binding is facilitated by the large contact area available for π-stacking between the aromatic residues of the peptide and the extended surfaces of graphene and the nanotube. The highly curved fullerene surface exhibits reduced efficiency for π-stacking but promotes increased peptide dynamics. We postulate that the increase in conformational dynamics of the amyloid peptide can be unfavorable for the formation of fibril competent structures. In contrast, extended fibril forming peptide conformations are promoted by the nanotube and graphene surfaces which can provide a template for fibril-growth. PMID:24339760

New horizons in mouse immunoinformatics: reliable in silico prediction of mouse class I histocompatibility major complex peptide binding affinity.

PubMed

Hattotuwagama, Channa K; Guan, Pingping; Doytchinova, Irini A; Flower, Darren R

2004-11-21

Quantitative structure-activity relationship (QSAR) analysis is a main cornerstone of modern informatic disciplines. Predictive computational models, based on QSAR technology, of peptide-major histocompatibility complex (MHC) binding affinity have now become a vital component of modern day computational immunovaccinology. Historically, such approaches have been built around semi-qualitative, classification methods, but these are now giving way to quantitative regression methods. The additive method, an established immunoinformatics technique for the quantitative prediction of peptide-protein affinity, was used here to identify the sequence dependence of peptide binding specificity for three mouse class I MHC alleles: H2-D(b), H2-K(b) and H2-K(k). As we show, in terms of reliability the resulting models represent a significant advance on existing methods. They can be used for the accurate prediction of T-cell epitopes and are freely available online ( http://www.jenner.ac.uk/MHCPred).

Strains of Actinomyces naeslundii and Actinomyces viscosus Exhibit Structurally Variant Fimbrial Subunit Proteins and Bind to Different Peptide Motifs in Salivary Proteins

PubMed Central

Li, Tong; Johansson, Ingegerd; Hay, Donald I.; Strömberg, Nicklas

1999-01-01

Oral strains of Actinomyces spp. express type 1 fimbriae, which are composed of major FimP subunits, and bind preferentially to salivary acidic proline-rich proteins (APRPs) or to statherin. We have mapped genetic differences in the fimP subunit genes and the peptide recognition motifs within the host proteins associated with these differential binding specificities. The fimP genes were amplified by PCR from Actinomyces viscosus ATCC 19246, with preferential binding to statherin, and from Actinomyces naeslundii LY7, P-1-K, and B-1-K, with preferential binding to APRPs. The fimP gene from the statherin-binding strain 19246 is novel and has about 80% nucleotide and amino acid sequence identity to the highly conserved fimP genes of the APRP-binding strains (about 98 to 99% sequence identity). The novel FimP protein contains an amino-terminal signal peptide, randomly distributed single-amino-acid substitutions, and structurally different segments and ends with a cell wall-anchoring and a membrane-spanning region. When agarose beads with CNBr-linked host determinant-specific decapeptides were used, A. viscosus 19246 bound to the Thr42Phe43 terminus of statherin and A. naeslundii LY7 bound to the Pro149Gln150 termini of APRPs. Furthermore, while the APRP-binding A. naeslundii strains originate from the human mouth, A. viscosus strains isolated from the oral cavity of rat and hamster hosts showed preferential binding to statherin and contained the novel fimP gene. Thus, A. viscosus and A. naeslundii display structurally variant fimP genes whose protein products are likely to interact with different peptide motifs and to determine animal host tropism. PMID:10225854

Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions.

PubMed

Pandalaneni, Sravan; Karuppiah, Vijaykumar; Saleem, Muhammad; Haynes, Lee P; Burgoyne, Robert D; Mayans, Olga; Derrick, Jeremy P; Lian, Lu-Yun

2015-07-24

Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca(2+)-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca(2+)/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca(2+)/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178-Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca(2+)/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178-Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions*

PubMed Central

Pandalaneni, Sravan; Karuppiah, Vijaykumar; Saleem, Muhammad; Haynes, Lee P.; Burgoyne, Robert D.; Mayans, Olga; Derrick, Jeremy P.; Lian, Lu-Yun

2015-01-01

Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca2+-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca2+/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca2+/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178–Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca2+/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178–Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1. PMID:25979333

An Exploration of the Calcium-Binding Mode of Egg White Peptide, Asp-His-Thr-Lys-Glu, and In Vitro Calcium Absorption Studies of Peptide-Calcium Complex.

PubMed

Sun, Na; Jin, Ziqi; Li, Dongmei; Yin, Hongjie; Lin, Songyi

2017-11-08

The binding mode between the pentapeptide (DHTKE) from egg white hydrolysates and calcium ions was elucidated upon its structural and thermodynamics characteristics. The present study demonstrated that the DHTKE peptide could spontaneously bind calcium with a 1:1 stoichiometry, and that the calcium-binding site corresponded to the carboxyl oxygen, amino nitrogen, and imidazole nitrogen atoms of the DHTKE peptide. Moreover, the effect of the DHTKE-calcium complex on improving the calcium absorption was investigated in vitro using Caco-2 cells. Results showed that the DHTKE-calcium complex could facilitate the calcium influx into the cytosol and further improve calcium absorption across Caco-2 cell monolayers by more than 7 times when compared to calcium-free control. This study facilitates the understanding about the binding mechanism between peptides and calcium ions as well as suggests a potential application of egg white peptides as nutraceuticals to improve calcium absorption.

Binding Modes of Phthalocyanines to Amyloid β Peptide and Their Effects on Amyloid Fibril Formation.

PubMed

Valiente-Gabioud, Ariel A; Riedel, Dietmar; Outeiro, Tiago F; Menacho-Márquez, Mauricio A; Griesinger, Christian; Fernández, Claudio O

2018-03-13

The inherent tendency of proteins to convert from their native states into amyloid aggregates is associated with a range of human disorders, including Alzheimer's and Parkinson's diseases. In that sense, the use of small molecules as probes for the structural and toxic mechanism related to amyloid aggregation has become an active area of research. Compared with other compounds, the structural and molecular basis behind the inhibitory interaction of phthalocyanine tetrasulfonate (PcTS) with proteins such as αS and tau has been well established, contributing to a better understanding of the amyloid aggregation process in these proteins. We present here the structural characterization of the binding of PcTS and its Cu(II) and Zn(II)-loaded forms to the amyloid β-peptide (Aβ) and the impact of these interactions on the peptide amyloid fibril assembly. Elucidation of the PcTS binding modes to Aβ 40 revealed the involvement of specific aromatic and hydrophobic interactions in the formation of the Aβ 40 -PcTS complex, ascribed to a binding mode in which the planarity and hydrophobicity of the aromatic ring system in the phthalocyanine act as main structural determinants for the interaction. Our results demonstrated that formation of the Aβ 40 -PcTS complex does not interfere with the progression of the peptide toward the formation of amyloid fibrils. On the other hand, conjugation of Zn(II) but not Cu(II) at the center of the PcTS macrocyclic ring modified substantially the binding profile of this phthalocyanine to Aβ 40 and became crucial to reverse the effects of metal-free PcTS on the fibril assembly of the peptide. Overall, our results provide a firm basis to understand the structural rules directing phthalocyanine-protein interactions and their implications on the amyloid fibril assembly of the target proteins; in particular, our results contradict the hypothesis that PcTS might have similar mechanisms of action in slowing the formation of a variety of

Synthesis of gold structures by gold-binding peptide governed by concentration of gold ion and peptide.

PubMed

Kim, Jungok; Kim, Dong-Hun; Lee, Sylvia J; Rheem, Youngwoo; Myung, Nosang V; Hur, Hor-Gil

2016-08-01

Although biological synthesis methods for the production of gold structures by microorganisms, plant extracts, proteins, and peptide have recently been introduced, there have been few reports pertaining to controlling their size and morphology. The gold ion and peptide concentrations affected on the size and uniformity of gold plates by a gold-binding peptide Midas-11. The higher concentration of gold ions produced a larger size of gold structures reached 125.5 μm, but an increased amount of Midas-11 produced a smaller size of gold platelets and increased the yield percentage of polygonal gold particles rather than platelets. The mechanisms governing factors controlling the production of gold structures were primarily related to nucleation and growth. These results indicate that the synthesis of gold architectures can be controlled by newly isolated and substituted peptides under different reaction conditions.

Calcitonin and Amylin Receptor Peptide Interaction Mechanisms: INSIGHTS INTO PEPTIDE-BINDING MODES AND ALLOSTERIC MODULATION OF THE CALCITONIN RECEPTOR BY RECEPTOR ACTIVITY-MODIFYING PROTEINS.

PubMed

Lee, Sang-Min; Hay, Debbie L; Pioszak, Augen A

2016-04-15

Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Down-regulation of poison ivy/oak-induced contact sensitivity by treatment with a class II MHC binding peptide:hapten conjugate.

PubMed

Gelber, C; Gemmell, L; McAteer, D; Homola, M; Swain, P; Liu, A; Wilson, K J; Gefter, M

1997-03-01

Immune regulation of contact sensitivity to the poison ivy/oak catechol was studied at the level of class II MHC-restricted T cell recognition of hapten:peptide conjugates. In this study we have shown that 1) T cells from C3H/HeN (H-2k) mice, immunized with a synthetic I-Ak binding peptide coupled to 3-pentadecyl-catechol (PDC; a representative catechol in urushiol), recognized peptides derived from syngeneic cells linked to the same catechol; 2) T cells from draining lymph nodes of C3H/HeN mice skin-painted with PDC proliferated in response to a peptide carrier:PDC conjugate only when it was linked at the 7th, but not the 4th or the 10th, position on the peptide carrier; and 3) tolerization studies confirmed down-regulation of PDC-induced delayed-type hypersensitivity following treatment with a single I-Ak binding peptide carrying PDC covalently bound to a lysine residue at the middle (7th) TCR contact position. Tolerization with peptide:PDC conjugate resulted in abrogation of hapten-specific T cell proliferative responses that correlated with diminished IL-2 secretion. On the basis of these data we propose that it may be sufficient to couple the hapten at a single, well-chosen position on a carrier peptide to target a relevant population of T cells involved in contact sensitivity.

PSBinder: A Web Service for Predicting Polystyrene Surface-Binding Peptides.

PubMed

Li, Ning; Kang, Juanjuan; Jiang, Lixu; He, Bifang; Lin, Hao; Huang, Jian

2017-01-01

Polystyrene surface-binding peptides (PSBPs) are useful as affinity tags to build a highly effective ELISA system. However, they are also a quite common type of target-unrelated peptides (TUPs) in the panning of phage-displayed random peptide library. As TUP, PSBP will mislead the analysis of panning results if not identified. Therefore, it is necessary to find a way to quickly and easily foretell if a peptide is likely to be a PSBP or not. In this paper, we describe PSBinder, a predictor based on SVM. To our knowledge, it is the first web server for predicting PSBP. The SVM model was built with the feature of optimized dipeptide composition and 87.02% (MCC = 0.74; AUC = 0.91) of peptides were correctly classified by fivefold cross-validation. PSBinder can be used to exclude highly possible PSBP from biopanning results or to find novel candidates for polystyrene affinity tags. Either way, it is valuable for biotechnology community.

Plasmin substrate binding site cooperativity guides the design of potent peptide aldehyde inhibitors.

PubMed

Swedberg, Joakim E; Harris, Jonathan M

2011-10-04

Perioperative bleeding is a cause of major blood loss and is associated with increased rates of postoperative morbidity and mortality. To combat this, antifibrinolytic inhibitors of the serine protease plasmin are commonly used to reduce bleeding during surgery. The most effective and previously widely used of these is the broad range serine protease inhibitor aprotinin. However, adverse clinical outcomes have led to use of alternative serine lysine analogues to inhibit plasmin. These compounds suffer from low selectivity and binding affinity. Consequently, a concerted effort to discover potent and selective plasmin inhibitors has developed. This study used a noncombinatorial peptide library to define plasmin's extended substrate specificity and guide the design of potent transition state analogue inhibitors. The various substrate binding sites of plasmin were found to exhibit a higher degree of cooperativity than had previously been appreciated. Peptide sequences capitalizing on these features produced high-affinity inhibitors of plasmin. The most potent of these, Lys-Met(sulfone)-Tyr-Arg-H [KM(O(2))YR-H], inhibited plasmin with a K(i) of 3.1 nM while maintaining 25-fold selectivity over plasma kallikrein. Furthermore, 125 nM (0.16 μg/mL) KM(O(2))YR-H attenuated fibrinolysis in vitro with an efficacy similar to that of 15 nM (0.20 μg/mL) aprotinin. To date, this is the most potent peptide inhibitor of plasmin that exhibits selectivity against plasma kallikrein, making this compound an attractive candidate for further therapeutic development.

DNA sensing by a Eu-binding peptide containing a proflavine unit.

PubMed

Ancel, Laetitia; Gateau, Christelle; Lebrun, Colette; Delangle, Pascale

2013-01-18

Synthesis of a lanthanide-binding peptide (LBP) for the detection of double-stranded DNA is presented. A proflavine moiety was introduced into a high affinity LBP involving two unnatural chelating amino acids in the Ln ion coordination. The Eu(3+)-LBP complex is demonstrated to bind to ct-DNA and to sensitize Eu luminescence. The DNA binding process is effectively detected via the Eu-centered luminescence thanks to the intimate coupling between the LBP scaffold and DNA intercalating unit.

Homology modelling of frequent HLA class-II alleles: A perspective to improve prediction of HLA binding peptide and understand the HLA associated disease susceptibility.

PubMed

Kashyap, Manju; Farooq, Umar; Jaiswal, Varun

2016-10-01

Human leukocyte antigen (HLA) plays significant role via the regulation of immune system and contribute in the progression and protection of many diseases. HLA molecules bind and present peptides to T- cell receptors which generate the immune response. HLA peptide interaction and molecular function of HLA molecule is the key to predict peptide binding and understanding its role in different diseases. The availability of accurate three dimensional (3D) structures is the initial step towards this direction. In the present work, homology modelling of important and frequent HLA-DRB1 alleles (07:01, 11:01 and 09:01) was done and acceptable models were generated. These modelled alleles were further refined and cross validated by using several methods including Ramachandran plot, Z-score, ERRAT analysis and root mean square deviation (RMSD) calculations. It is known that numbers of allelic variants are related to the susceptibility or protection of various infectious diseases. Difference in amino acid sequences and structures of alleles were also studied to understand the association of HLA with disease susceptibility and protection. Susceptible alleles showed more amino acid variations than protective alleles in three selected diseases caused by different pathogens. Amino acid variations at binding site were found to be more than other part of alleles. RMSD values were also higher at variable positions within binding site. Higher RMSD values indicate that mutations occurring at peptide binding site alter protein structure more than rest of the protein. Hence, these findings and modelled structures can be used to design HLA-DRB1 binding peptides to overcome low prediction accuracy of HLA class II binding peptides. Furthermore, it may help to understand the allele specific molecular mechanisms involved in susceptibility/resistance against pathogenic diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

NetMHCpan, a Method for Quantitative Predictions of Peptide Binding to Any HLA-A and -B Locus Protein of Known Sequence

PubMed Central

Nielsen, Morten; Lundegaard, Claus; Blicher, Thomas; Lamberth, Kasper; Harndahl, Mikkel; Justesen, Sune; Røder, Gustav; Peters, Bjoern; Sette, Alessandro; Lund, Ole; Buus, Søren

2007-01-01

Background Binding of peptides to Major Histocompatibility Complex (MHC) molecules is the single most selective step in the recognition of pathogens by the cellular immune system. The human MHC class I system (HLA-I) is extremely polymorphic. The number of registered HLA-I molecules has now surpassed 1500. Characterizing the specificity of each separately would be a major undertaking. Principal Findings Here, we have drawn on a large database of known peptide-HLA-I interactions to develop a bioinformatics method, which takes both peptide and HLA sequence information into account, and generates quantitative predictions of the affinity of any peptide-HLA-I interaction. Prospective experimental validation of peptides predicted to bind to previously untested HLA-I molecules, cross-validation, and retrospective prediction of known HIV immune epitopes and endogenous presented peptides, all successfully validate this method. We further demonstrate that the method can be applied to perform a clustering analysis of MHC specificities and suggest using this clustering to select particularly informative novel MHC molecules for future biochemical and functional analysis. Conclusions Encompassing all HLA molecules, this high-throughput computational method lends itself to epitope searches that are not only genome- and pathogen-wide, but also HLA-wide. Thus, it offers a truly global analysis of immune responses supporting rational development of vaccines and immunotherapy. It also promises to provide new basic insights into HLA structure-function relationships. The method is available at http://www.cbs.dtu.dk/services/NetMHCpan. PMID:17726526

SABinder: A Web Service for Predicting Streptavidin-Binding Peptides.

PubMed

He, Bifang; Kang, Juanjuan; Ru, Beibei; Ding, Hui; Zhou, Peng; Huang, Jian

2016-01-01

Streptavidin is sometimes used as the intended target to screen phage-displayed combinatorial peptide libraries for streptavidin-binding peptides (SBPs). More often in the biopanning system, however, streptavidin is just a commonly used anchoring molecule that can efficiently capture the biotinylated target. In this case, SBPs creeping into the biopanning results are not desired binders but target-unrelated peptides (TUP). Taking them as intended binders may mislead subsequent studies. Therefore, it is important to find if a peptide is likely to be an SBP when streptavidin is either the intended target or just the anchoring molecule. In this paper, we describe an SVM-based ensemble predictor called SABinder. It is the first predictor for SBP. The model was built with the feature of optimized dipeptide composition. It was observed that 89.20% (MCC = 0.78; AUC = 0.93; permutation test, p < 0.001) of peptides were correctly classified. As a web server, SABinder is freely accessible. The tool provides a highly efficient way to exclude potential SBP when they are TUP or to facilitate identification of possibly new SBP when they are the desired binders. In either case, it will be helpful and can benefit related scientific community.

Competitive binding effects on surface-enhanced Raman scattering of peptide molecules

NASA Astrophysics Data System (ADS)

Seballos, Leo; Richards, Nicole; Stevens, Daniel J.; Patel, Mira; Kapitzky, Laura; Lokey, Scott; Millhauser, Glenn; Zhang, Jin Z.

2007-10-01

Surface enhanced Raman scattering (SERS) has been conducted on tryptophan (W), proline (P) and tyrosine (Y) containing peptides that include W-P-Y, Y-P-W, W-P-P-P-Y, Y-P-P-P-W, W-P-P-P-P-P-Y, and Y-P-P-P-P-P-W to gain insight into molecular binding behavior on a metal substrate to eventually apply in protein SERS detection. The peptides are shown to bind through the molecule's carboxylic end, but the strong affinity of the tryptophan residue to the substrate surface, in conjunction with its large polarizability, dominates each molecule's SERS signal with the strong presence of its ring modes in all samples. These results are important for understanding SERS of protein molecules.

Rate and extent of protein localization is controlled by peptide-binding domain association kinetics and morphology.

PubMed

Mills, Evan; Truong, Kevin

2009-06-01

Protein localization is an important regulatory mechanism in many cell signaling pathways such as cytoskeletal organization and genetic regulation. The specific mechanism of protein localization determines the kinetics and morphological constraints of protein translocation, and thus affects the rate and extent of localization. To investigate the affect of localization kinetics and morphology on protein localization, we designed a protein localization system based on Ca(2+)-calmodulin and Src homology 3 domain binding peptides that can translocate between specific localizations in response to a Ca(2+) signal. We used a stochastic biomolecular simulator to predict that such a protein localization system will exhibit slower and less complete translocations when the association kinetics of a binding domain and peptide are reduced. As well, we predicted that increasing the diffusion resistance by manipulating the morphology of the system would similarly impair translocation speed and completeness. We then constructed a network of synthetic fusion proteins and showed that these predictions could be qualitatively confirmed in vitro. This work provides a basis for explaining the different characteristics (rate and extent) of protein transport and localization in cells as a consequence of the kinetics and morphology of the transport mechanism.

A 31-residue peptide induces aggregation of tau's microtubule-binding region in cells

NASA Astrophysics Data System (ADS)

Stöhr, Jan; Wu, Haifan; Nick, Mimi; Wu, Yibing; Bhate, Manasi; Condello, Carlo; Johnson, Noah; Rodgers, Jeffrey; Lemmin, Thomas; Acharya, Srabasti; Becker, Julia; Robinson, Kathleen; Kelly, Mark J. S.; Gai, Feng; Stubbs, Gerald; Prusiner, Stanley B.; Degrado, William F.

2017-09-01

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer's. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behaviour of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fibre diffraction, hydrogen-deuterium exchange and solid-state NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide.

Clicked bis-PEG-peptide conjugates for studying calmodulin-Kv7.2 channel binding.

PubMed

Bonache, M Angeles; Alaimo, Alessandro; Malo, Covadonga; Millet, Oscar; Villarroel, Alvaro; González-Muñiz, Rosario

2014-11-28

The recombinant Kv7.2 calmodulin (CaM) binding site (Q2AB CaMBD) shows a high tendency to aggregate, thus complicating biochemical and structural studies. To facilitate these studies we have conceived bis-PEG-peptide CaMBD-mimetics linking helices A and B in single, easy to handle molecules. Short PEG chains were selected as spacers between the two peptide molecules, and a Cu(i)-catalyzed cycloaddition (CuAAC) protocol was used to assemble the final bis-PEG-peptide conjugate, by the convenient functionalization of PEG arms with azide and alkyne groups. The resulting conjugates, with a certain helical character in TFE solutions (CD), showed nanomolar affinity in a fluorescence CaM binding in vitro assay, higher than just the sum of the precursor PEG-peptide affinities, thus validating our design. The approach to these first described examples of Kv7.2 CaMBD-mimetics could pave the way to chimeric conjugates merging helices A and B from different Kv7 subunits.

Specific material recognition by small peptides mediated by the interfacial solvent structure.

PubMed

Schneider, Julian; Ciacchi, Lucio Colombi

2012-02-01

We present evidence that specific material recognition by small peptides is governed by local solvent density variations at solid/liquid interfaces, sensed by the side-chain residues with atomic-scale precision. In particular, we unveil the origin of the selectivity of the binding motif RKLPDA for Ti over Si using a combination of metadynamics and steered molecular dynamics simulations, obtaining adsorption free energies and adhesion forces in quantitative agreement with corresponding experiments. For an accurate description, we employ realistic models of the natively oxidized surfaces which go beyond the commonly used perfect crystal surfaces. These results have profound implications for nanotechnology and materials science applications, offering a previously missing structure-function relationship for the rational design of materials-selective peptide sequences. © 2011 American Chemical Society

A Blocking Group Scan Using a Spherical Organometallic Complex Identifies an Unprecedented Binding Mode with Potent Activity In Vitro and In Vivo for the Opioid Peptide Dermorphin.

PubMed

Strack, Martin; Bedini, Andrea; Yip, King T; Lombardi, Sara; Siegmund, Daniel; Stoll, Raphael; Spampinato, Santi M; Metzler-Nolte, Nils

2016-10-04

Herein, the selective enforcement of one particular receptor-ligand interaction between specific domains of the μ-selective opioid peptide dermorphin and the μ opioid receptor is presented. For this, a blocking group scan is described which exploits the steric demand of a bis(quinolinylmethyl)amine rhenium(I) tricarbonyl complex conjugated to a number of different, strategically chosen positions of dermorphin. The prepared peptide conjugates lead to the discovery of two different binding modes: An expected N-terminal binding mode corresponds to the established view of opioid peptide binding, whereas an unexpected C-terminal binding mode is newly discovered. Surprisingly, both binding modes provide high affinity and agonistic activity at the μ opioid receptor in vitro. Furthermore, the unprecedented C-terminal binding mode shows potent dose-dependent antinociception in vivo. Finally, in silico docking studies support receptor activation by both dermorphin binding modes and suggest a biological relevance for dermorphin itself. Relevant ligand-protein interactions are similar for both binding modes, which is in line with previous protein mutation studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of iron-binding activity of collagen peptides prepared from the scales of four cultivated fishes in Taiwan.

PubMed

Huang, Chun-Yung; Wu, Chien-Hui; Yang, Jing-Iong; Li, Ying-Han; Kuo, Jen-Min

2015-12-01

Iron deficiency is one of the most concerning deficiency problems in the world. It may generate several adverse effects such as iron deficiency anemia (IDA) and reduced physical and intellectual working capacity. The aim of this study is to evaluate the Fe(II)-binding activity of collagen peptides from fishery by-products. Lates calcarifer, Mugil cephalus, Chanos chanos, and Oreochromis spp are four major cultivated fishes in Taiwan; thousands of scales of these fish are wasted without valuable utilization. In this study, scales of these fish were hydrolyzed by papain plus flavourzyme. Collagen peptides were obtained and compared for their Fe(II)-binding activity. Collagen peptides from Chanos chanos showed the highest Fe(II)-binding activity, followed by those from Lates calcarifer and Mugil cephalus; that from Oreochromis spp exhibited the lowest one. Fe(II)-binding activity of collagen peptides from fish scales was also confirmed with a dialysis method. Molecular weight (MW) distributions of the collagen peptides from scales of four fish are all < 10 kDa, and averaged 1.3 kDa. Hydrolysates of fish scales were further partially purified with ion exchange chromatography. Fractions having Fe(II)-binding activity were obtained and their activity compared. Data obtained showed that collagen peptides from fish scales did have Fe(II)-binding activity. This is the first observation elucidating fish scale collagen possessing this functionality. The results from this study also indicated that collagen peptides from fish scales could be applied in industry as a bioresource. Copyright © 2014. Published by Elsevier B.V.

MUSI: an integrated system for identifying multiple specificity from very large peptide or nucleic acid data sets.

PubMed

Kim, Taehyung; Tyndel, Marc S; Huang, Haiming; Sidhu, Sachdev S; Bader, Gary D; Gfeller, David; Kim, Philip M

2012-03-01

Peptide recognition domains and transcription factors play crucial roles in cellular signaling. They bind linear stretches of amino acids or nucleotides, respectively, with high specificity. Experimental techniques that assess the binding specificity of these domains, such as microarrays or phage display, can retrieve thousands of distinct ligands, providing detailed insight into binding specificity. In particular, the advent of next-generation sequencing has recently increased the throughput of such methods by several orders of magnitude. These advances have helped reveal the presence of distinct binding specificity classes that co-exist within a set of ligands interacting with the same target. Here, we introduce a software system called MUSI that can rapidly analyze very large data sets of binding sequences to determine the relevant binding specificity patterns. Our pipeline provides two major advances. First, it can detect previously unrecognized multiple specificity patterns in any data set. Second, it offers integrated processing of very large data sets from next-generation sequencing machines. The results are visualized as multiple sequence logos describing the different binding preferences of the protein under investigation. We demonstrate the performance of MUSI by analyzing recent phage display data for human SH3 domains as well as microarray data for mouse transcription factors.

Hydrogen/Deuterium Exchange Reflects Binding of Human Centrin 2 to Ca2+ and Xeroderma Pigmentosum Group C Peptide: An Example of EX1 Kinetics

PubMed Central

Sperry, Justin B.; Ryan, Zachary C.; Kumar, Rajiv; Gross, Michael L.

2012-01-01

Xeroderma pigmentosum (XP) is a genetic disease affecting 1 in 10,000-100,000 and predisposes people to early-age skin cancer, a disease that is increasing. Those with XP have decreased ability to repair UV-induced DNA damage, leading to increased susceptibility of cancerous non-melanomas and melanomas. A vital, heterotrimeric protein complex is linked to the nucleotide excision repair pathway for the damaged DNA. The complex consists of XPC protein, human centrin 2, and RAD23B. One of the members, human centrin 2, is a ubiquitous, acidic, Ca2+-binding protein belonging to the calmodulin superfamily. The XPC protein contains a sequence motif specific for binding to human centrin 2. We report here the Ca2+-binding properties of human centrin 2 and its interaction with the XPC peptide motif. We utilized a region-specific H/D exchange protocol to localize the interaction of the XPC peptide with the C-terminal domain of centrin, the binding of which is different than that of calmodulin complexes. The binding dynamics of human centrin 2 to the XPC peptide in the absence and presence of Ca2+ are revealed by the observation of EX1 H/D exchange regime, indicating that a locally unfolded population exists in solution and undergoes fast H/D exchange. PMID:23439742

Peptide-bacteria interactions using engineered surface-immobilized peptides from class IIa bacteriocins.

PubMed

Etayash, Hashem; Norman, Lana; Thundat, Thomas; Kaur, Kamaljit

2013-03-26

Specificity of the class IIa bacteriocin Leucocin A (LeuA), an antimicrobial peptide active against Gram-positive bacteria, including Listeria monocytogenes , is known to be dictated by the C-terminal amphipathic helical region, including the extended hairpin-like structure. However, its specificity when attached to a substrate has not been investigated. Exploiting properties of LeuA, we have synthesized two LeuA derivatives, which span the amphipathic helical region of the wild-type LeuA, consisting of 14- (14AA LeuA, CWGEAFSAGVHRLA) and 24-amino acid residues (24AA LeuA, CSVNWGEAFSAGVHRLANGGNGFW). The peptides were purified to >95% purity, as shown by analytical RP-HPLC and mass spectrometry. By including an N-terminal cysteine group, the tailored peptide fragments were readily immobilized at the gold interfaces. The resulting thickness and molecular orientation, determined by ellipsometry and grazing angle infrared spectroscopy, respectively, indicated that the peptides were covalently immobilized in a random helical orientation. The bacterial specificity of the anchored peptide fragments was tested against Gram-positive and Gram-negative bacteria. Our results showed that the adsorbed 14AA LeuA exhibited no specificity toward the bacterial strains, whereas the surface-immobilized 24AA LeuA displayed significant binding toward Gram-positive bacteria with various binding affinities from one strain to another. The 14AA LeuA did not show binding as this fragment is most likely too short in length for recognition by the membrane-bound receptor on the target bacterial cell membrane. These results support the potential use of class IIa bacteriocins as molecular recognition elements in biosensing platforms.

Binding Properties of a Peptide Derived from β-Lactamase Inhibitory Protein

PubMed Central

Rudgers, Gary W.; Huang, Wanzhi; Palzkill, Timothy

2001-01-01

To overcome the antibiotic resistance mechanism mediated by β-lactamases, small-molecule β-lactamase inhibitors, such as clavulanic acid, have been used. This approach, however, has applied selective pressure for mutations that result in β-lactamases no longer sensitive to β-lactamase inhibitors. On the basis of the structure of β-lactamase inhibitor protein (BLIP), novel peptide inhibitors of β-lactamase have been constructed. BLIP is a 165-amino-acid protein that is a potent inhibitor of TEM-1 β-lactamase (Ki = 0.3 nM). The cocrystal structure of TEM-1 β-lactamase and BLIP indicates that residues 46 to 51 of BLIP make critical interactions with the active site of TEM-1 β-lactamase. A peptide containing this six-residue region of BLIP was found to retain sufficient binding energy to interact with TEM-1 β-lactamase. Inhibition assays with the BLIP peptide reveal that, in addition to inhibiting TEM-1 β-lactamase, the peptide also inhibits a class A β-lactamase and a class C β-lactamase that are not inhibited by BLIP. The crystal structures of class A and C β-lactamases and two penicillin-binding proteins (PBPs) reveal that the enzymes have similar three-dimensional structures in the vicinity of the active site. This similarity suggests that the BLIP peptide inhibitor may have a broad range of activity that can be used to develop novel small-molecule inhibitors of various classes of β-lactamases and PBPs. PMID:11709298

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

PubMed

Deuerling, Elke; Patzelt, Holger; Vorderwülbecke, Sonja; Rauch, Thomas; Kramer, Günter; Schaffitzel, Elke; Mogk, Axel; Schulze-Specking, Agnes; Langen, Hanno; Bukau, Bernd

2003-03-01

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, deltatig, 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 degrees 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 deltatig 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.

Specific peptide for functionalization of GaN

NASA Astrophysics Data System (ADS)

Estephan, E.; Larroque, C.; Cloitre, T.; Cuisinier, F. J. G.; Gergely, C.

2008-04-01

Nanobiotechnology aims to exploit biomolecular recognition and self-assembly capabilities for integrating advanced materials into medicine and biology. However frequent problems are encountered at the interface of substrate-biological molecule, as the direct physical adsorption of biological molecules is dependent of unpredictable non-specific interactions with the surface, often causing their denaturation. Therefore, a proper functionalization of the substrate should avoid a loss of biological activity. In this work we address the functionalization of the semiconductor GaN (0001) for biosensing applications. The basic interest of using III-V class semiconductors is their good light emitting properties and a fair chemical stability that allows various applications of these materials. The technology chosen to elaborate GaN-specific peptides is the combinatorial phage-display method, a biological screening procedure based on affinity selection. An M13 bacteriophage library has been used to screen 10 10 different peptides against the GaN (0001) surface to finally isolate one specific peptide. The preferential attachment of the biotinylated selected peptide onto the GaN (0001), in close proximity to a surface of different chemical and structural composition has been demonstrated by fluorescence microscopy. Further physicochemical studies have been initiated to evaluate the semiconductor-peptide interface and understand the details in the specific recognition of peptides for semiconductor substrates. Fourier Transform Infrared spectroscopy in Attenuated Total Reflection mode (FTIR-ATR) has been employed to prove the presence of peptides on the surface. Our Atomic Force Microscopy (AFM) studies on the morphology of the GaN surface after functionalization revealed a total surface coverage by a very thin, homogeneous peptide layer. Due to its good biocompatibility, functionalized GaN devices might evolve in a new class of implantable biosensors for medical applications.

Identification of an Electrostatic Ruler Motif for Sequence-Specific Binding of Collagenase to Collagen.

PubMed

Subramanian, Sundar Raman; Singam, Ettayapuram Ramaprasad Azhagiya; Berinski, Michael; Subramanian, Venkatesan; Wade, Rebecca C

2016-08-25

Sequence-specific cleavage of collagen by mammalian collagenase plays a pivotal role in cell function. Collagenases are matrix metalloproteinases that cleave the peptide bond at a specific position on fibrillar collagen. The collagenase Hemopexin-like (HPX) domain has been proposed to be responsible for substrate recognition, but the mechanism by which collagenases identify the cleavage site on fibrillar collagen is not clearly understood. In this study, Brownian dynamics simulations coupled with atomic-detail and coarse-grained molecular dynamics simulations were performed to dock matrix metalloproteinase-1 (MMP-1) on a collagen IIIα1 triple helical peptide. We find that the HPX domain recognizes the collagen triple helix at a conserved R-X11-R motif C-terminal to the cleavage site to which the HPX domain of collagen is guided electrostatically. The binding of the HPX domain between the two arginine residues is energetically stabilized by hydrophobic contacts with collagen. From the simulations and analysis of the sequences and structural flexibility of collagen and collagenase, a mechanistic scheme by which MMP-1 can recognize and bind collagen for proteolysis is proposed.

Targeted Delivery of an Antigenic Peptide to the Endoplasmic Reticulum: Application for Development of a Peptide Therapy for Ankylosing Spondylitis

PubMed Central

Yu, Hui-Chun; Lu, Ming-Chi; Li, Chin; Huang, Hsien-Lu; Huang, Kuang-Yung; Liu, Su-Qin; Lai, Ning-Sheng; Huang, Hsien-Bin

2013-01-01

The development of suitable methods to deliver peptides specifically to the endoplasmic reticulum (ER) can provide some potential therapeutic applications of such peptides. Ankylosing spondylitis (AS) is strongly associated with the expression of human leukocytic antigen-B27 (HLA-B27). HLA-B27 heavy chain (HC) has a propensity to fold slowly resulting in the accumulation of misfolded HLA-B27 HC in the ER, triggering the unfolded protein response, and forming a homodimer, (B27-HC)2. Natural killer cells and T-helper 17 cells are then activated, contributing to the major pathogenic potentials of AS. The HLA-B27 HC is thus an important target, and delivery of an HLA-B27-binding peptide to the ER capable of promoting HLA-B27 HC folding is a potential mechanism for AS therapy. Here, we demonstrate that a His6-ubiquitin-tagged Tat-derived peptide (THU) can deliver an HLA-B27-binding peptide to the ER promoting HLA-B27 HC folding. The THU-HLA-B27-binding peptide fusion protein crossed the cell membrane to the cytosol through the Tat-derived peptide. The HLA-B27-binding peptide was specifically cleaved from THU by cytosolic ubiquitin C-terminal hydrolases and subsequently transported into the ER by the transporter associated with antigen processing. This approach has potential application in the development of peptide therapy for AS. PMID:24155957

Selection of peptides for binding semiconductor and magnetic materials for the purpose of organizing nanoscaled materials

NASA Astrophysics Data System (ADS)

Whaley, Sandra Renee

A peptide combinatorial approach, also known as phage display, was used to isolate peptides with the ability to bind semiconductor (GaAs, GaN, and InP) and magnetic (Fe2O3 and Fe3O4) materials. The commercially available combinatorial libraries contain randomized peptides either twelve (Ph.D-12(TM)) or seven (Ph.D-C7C(TM)) amino acids in length. The peptides are displayed on the pIII protein of M13 bacteriophage, which have been imaged by atomic force microscopy and transmission electron microscopy. After seven rounds of phage selection with a constrained seven amino acid sequence library (Ph.D-C7C(TM)), two sequences were isolated for binding Fe3O4 (MG-127 and MG-78). The haematite surface was screened with the same library and four unique sequences were isolated after six rounds of selection (HM-95, HM-101, HM-103, and HM-111). According to binding experiments (MG-78 v. MG-127 on Fe3O 4, MG-127 v. HM-95 on Fe3O4 and Fe2O 3, and MG-127 v. HM-95 on gamma-Fe2O3), the MG-127 clone had the highest affinity for iron oxide surfaces (magnetite, haematite, and maghemite) among the clones tested. The Fe3O 4 clone MG-127 displayed the ability to organize Fe3O 4 nanoparticles along bundles of phage. The synthetic peptide analog of this clone was used in the organization of nanoparticles onto the surface of latex beads. The surfaces of the III-V semiconductors were studied using x-ray photoelectron spectroscopy to determine their reactivity in the aqueous conditions used for phage selection. The GaN surface was shown to oxidize the least under these conditions, aiding in the ability to isolate a consensus amino acid sequence responsible for binding to this surface. The G1-3 clone isolated for binding the GaAs (100) surface displayed preferential binding to the GaAs (100) surface over Si (100), GaAs (111) A, GaAs (111) B, and AlGaAs. The synthetic peptide analog of the G12-3 clone was found to preferentially bind to GaAs (100) over either GaAs (111) surfaces or InP (100). This

Rat MHC-linked peptide transporter alleles strongly influence peptide binding by HLA-B27 but not B27-associated inflammatory disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simmons, W.A.; Satumtira, Nimman; Taurog, J.D.

1996-02-15

Rats transgenic for the human MHC molecule HLA-B27 were used to study the effect of two alleles, cim{sup a} and cim{sup b}, which are associated with peptide transport by the MHC-encoded Tap2 transporter, on the function of HLA-B27 as a restriction element for CTL recognition of the male H-Y minor H Ag and on the multisystem inflammatory disease characteristic of B27 transgenic rats. Anti-H-Y CTL generated in cim{sup a} B27 transgenic rats lysed male B27 cim{sup b/b} targets significantly less well than cim{sup a/a} or cim{sup a/b} targets. Addition of exogenous H-Y peptides to male B27 cim{sup b/b} targets increasedmore » susceptibility to lysis to the level of cim{sup a/a} targets sensitized with exogenous H-Y peptides. {sup 3}H-labeled peptides eluted from B27 molecules of lymphoblasts from rats of two cim{sup b} and three cim{sup a} RT1 haplotypes showed that the cim{sup b} peptide pool favors comparatively longer and/or more hydrophobic peptides. These results indicate that RT1-linked Tap2 polymorphism in the rat strongly influences peptide loading of HLA-B27. Nonetheless, the prevalence and severity of multisystem inflammatory lesions were comparable in backcross rats bearing either cim{sup a/b} or cim{sup b/b}. It thus appears either that binding of specific peptides to B27 is unimportant in the pathogenesis of B27-associated disease or that the critical peptides, unlike H-Y and many others, are not influenced by Tap transporter polymorphism. 42 refs., 6 figs., 3 tabs.« less

Protection of Nonself Surfaces from Complement Attack by Factor H-Binding Peptides: Implications for Therapeutic Medicine

PubMed Central

Wu, You-Qiang; Qu, Hongchang; Sfyroera, Georgia; Tzekou, Apostolia; Kay, Brian K.; Nilsson, Bo; Ekdahl, Kristina Nilsson; Ricklin, Daniel; Lambris, John D.

2011-01-01

Exposure of nonself surfaces such as those of biomaterials or transplanted cells and organs to host blood frequently triggers innate immune responses, thereby affecting both their functionality and tolerability. Activation of the alternative pathway of complement plays a decisive role in this unfavorable reaction. Whereas previous studies demonstrated that immobilization of physiological regulators of complement activation (RCA) can attenuate this foreign body-induced activation, simple and efficient approaches for coating artificial surfaces with intact RCA are still missing. The conjugation of small molecular entities that capture RCA with high affinity is an intriguing alternative, as this creates a surface with autoregulatory activity upon exposure to blood. We therefore screened two variable cysteine-constrained phage-displayed peptide libraries for factor H-binding peptides. We discovered three peptide classes that differed with respect to their main target binding areas. Peptides binding to the broad middle region of factor H (domains 5–18) were of particular interest, as they do not interfere with either regulatory or binding activities. One peptide in this group (5C6) was further characterized and showed high factor H-capturing activity while retaining its functional integrity. Most importantly, when 5C6 was coated to a model polystyrene surface and exposed to human lepirudin-anticoagulated plasma, the bound peptide captured factor H and substantially inhibited complement activation by the alternative pathway. Our study therefore provides a promising and novel approach to produce therapeutic materials with enhanced biocompatibility. PMID:21339361

M13 phage peptide ZL4 exerts its targeted binding effect on schistosoma japonicum via alkaline phosphatase.

PubMed

Liu, Yan; Yang, Shenghui; Xiao, Jianhua; Yu, Liang; Chen, Li; Zou, Ju; Wang, Kegeng; Tan, Sijie; Yu, Zhengyang; Zeng, Qingren

2015-01-01

The present study was to determine the targeting effect of M13 phage peptide ZL4 (MppZL4) on Schistosoma japonicum (S.j). Mice infected with S.j were injected with MppZL4. Real-time PCR was used to detect the distribution and metabolism of MppZL4 in the livers and lungs of mice. In vivo refusion test was performed to detect the targeting of MppZL4. Western blotting was employed to determine the expression of MppZL4. Live imaging was used to detect the distribution of oligopeptide MppZL4. Immunohistochemistry was employed to determine MppZL4 location on adult S.j body surface. Gomori method was employed to detect the influence of oligopeptide MppZL4 on alkaline phosphatase activity. The distribution and metabolism of MppZL4 and M13KE are not significantly different from each other at each time point. The abundance of MppZL4 is changed as S.j migrates in mice. The targeted binding effect of MppZL4 varies at different stages. ZL4 oligopeptide targets S.j in mice. The specific binding sites of MppZL4 on S.j body are mainly located in syncytial cells. The binding sites of MppZL4 on S.j body surface might be ALP or ALP-related proteins. MppZL4 had targeted binding effect on S.j with its binding site being associated with proteins related to S.j alkaline phosphatase. S.j tegument had a specifically binding site with exogenous peptides, offering new means to explore the interactions between hosts and parasites. Additionally, MppZL4 can possibly be used as targeting molecules in worm-resistant drugs or as tracing molecules in imaging diagnosis technologies.

Hydrophobic Peptides Affect Binding of Calmodulin and Ca2+ as Explored by H/D Amide Exchange and Mass Spectrometry

PubMed Central

Sperry, Justin B.; Huang, Richard Y-C.; Zhu, Mei M.; Rempel, Don L.; Gross, Michael L.

2010-01-01

Calmodulin (CaM), a ubiquitous intracellular sensor protein, binds Ca2+ and interacts with various targets as part of signal transduction. Using hydrogen/deuterium exchange (H/DX) and a high resolution PLIMSTEX (Protein-Ligand Interactions by Mass Spectrometry, Titration, and H/D Exchange) protocol, we examined five different states of calmodulin: calcium-free, calcium-loaded, and three states of calcium-loaded in the presence of either melittin, mastoparan, or skeletal myosin light-chain kinase (MLCK). When CaM binds Ca2+, the extent of HDX decreased, consistent with the protein becoming stabilized upon binding. Furthermore, Ca2+-saturated calmodulin exhibits increased protection when bound to the peptides, forming high affinity complexes. The protocol reveals significant changes in EF hands 1, 3, and 4 with saturating levels of Ca2+. Titration of the protein using PLIMSTEX provides the binding affinity of Ca2+ to calmodulin within previously reported values. The affinities of calmodulin to Ca2+ increase by factors of 300 and 1000 in the presence of melittin and mastoparan, respectively. A modified PLIMSTEX protocol whereby the protein is digested to component peptides gives a region-specific titration. The titration data taken in this way show a decrease in the root mean square fit of the residuals, indicating a better fit of the data. The global H/D exchange results and those obtained in a region-specific way provide new insight into the Ca2+-binding properties of this well-studied protein. PMID:21765646

Peptide aptamers: The versatile role of specific protein function inhibitors in plant biotechnology.

PubMed

Colombo, Monica; Mizzotti, Chiara; Masiero, Simona; Kater, Martin M; Pesaresi, Paolo

2015-11-01

In recent years, peptide aptamers have emerged as novel molecular tools that have attracted the attention of researchers in various fields of basic and applied science, ranging from medicine to analytical chemistry. These artificial short peptides are able to specifically bind, track, and inhibit a given target molecule with high affinity, even molecules with poor immunogenicity or high toxicity, and represent a remarkable alternative to antibodies in many different applications. Their use is on the rise, driven mainly by the medical and pharmaceutical sector. Here we discuss the enormous potential of peptide aptamers in both basic and applied aspects of plant biotechnology and food safety. The different peptide aptamer selection methods available both in vivo and in vitro are introduced, and the most important possible applications in plant biotechnology are illustrated. In particular, we discuss the generation of broad-based virus resistance in crops, "reverse genetics" and aptasensors in bioassays for detecting contaminations in food and feed. Furthermore, we suggest an alternative to the transfer of peptide aptamers into plant cells via genetic transformation, based on the use of cell-penetrating peptides that overcome the limits imposed by both crop transformation and Genetically Modified Organism commercialization. © 2015 Institute of Botany, Chinese Academy of Sciences.

Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

PubMed Central

Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Delangle, Pascale; Guilloreau, Luc; Adriano, Jean-Marc; Berthomieu, Catherine

2012-01-01

To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. PMID:22870263

On the composition of the preimmune repertoire of T cells specific for Peptide-major histocompatibility complex ligands.

PubMed

Jenkins, Marc K; Chu, H Hamlet; McLachlan, James B; Moon, James J

2010-01-01

Millions of T cells are produced in the thymus, each expressing a unique alpha/beta T cell receptor (TCR) capable of binding to a foreign peptide in the binding groove of a host major histocompatibility complex (MHC) molecule. T cell-mediated immunity to infection is due to the proliferation and differentiation of rare clones in the preimmune repertoire that by chance express TCRs specific for peptide-MHC (pMHC) ligands derived from the microorganism. Here we review recent findings that have altered our understanding of how the preimmune repertoire is established. Recent structural studies indicate that a germline-encoded tendency of TCRs to bind MHC molecules contributes to the MHC bias of T cell repertoires. It has also become clear that the preimmune repertoire contains functionally heterogeneous subsets including recent thymic emigrants, mature naive phenotype cells, memory phenotype cells, and natural regulatory T cells. In addition, sensitive new detection methods have revealed that the repertoire of naive phenotype T cells consists of distinct pMHC-specific populations that consistently vary in size in different individuals. The implications of these new findings for the clonal selection theory, self-tolerance, and immunodominance are discussed.

Screening of a library of T7 phage-displayed peptides identifies alphaC helix in 14-3-3 protein as a CBP501-binding site.

PubMed

Matsumoto, Yuki; Shindo, Yosuke; Takakusagi, Yoichi; Takakusagi, Kaori; Tsukuda, Senko; Kusayanagi, Tomoe; Sato, Hitoshi; Kawabe, Takumi; Sugawara, Fumio; Sakaguchi, Kengo

2011-12-01

CBP501 is a chemically modified peptide composed of twelve unnatural d-amino acids, which inhibits Chk kinase and abrogates G2 arrest induced by DNA-damaging agents. Here we identified an alphaC helix in 14-3-3 protein as a CBP501-binding site using T7 phage display technology. An affinity selection of T7 phage-displayed peptide using biotinylated CBP501 identified a 14-mer peptide NSDCIISRKIEQKE. This peptide sequence showed similarity to a portion of the alphaC helix of human 14-3-3ε, suggesting that CBP501 may bind to this region. Surface plasmon resonance (SPR) and ELISA demonstrated that CBP501 interacts with 14-3-3ε specifically at the screen-guided region. An avidin-agarose bead pull-down assay showed that CBP501 also binds to other 14-3-3 isoforms in Jurkat cells. Among the other known Chk kinase inhibitors tested, CBP501 showed the strongest affinity for 14-3-3ε. Thus, we conclude that in addition to the direct inhibition of Chk kinase activity, CBP501 directly binds to cellular 14-3-3 proteins through alphaC helix. Copyright © 2011 Elsevier Ltd. All rights reserved.

A community resource benchmarking predictions of peptide binding to MHC-I molecules.

PubMed

Peters, Bjoern; Bui, Huynh-Hoa; Frankild, Sune; Nielson, Morten; Lundegaard, Claus; Kostem, Emrah; Basch, Derek; Lamberth, Kasper; Harndahl, Mikkel; Fleri, Ward; Wilson, Stephen S; Sidney, John; Lund, Ole; Buus, Soren; Sette, Alessandro

2006-06-09

Recognition of peptides bound to major histocompatibility complex (MHC) class I molecules by T lymphocytes is an essential part of immune surveillance. Each MHC allele has a characteristic peptide binding preference, which can be captured in prediction algorithms, allowing for the rapid scan of entire pathogen proteomes for peptide likely to bind MHC. Here we make public a large set of 48,828 quantitative peptide-binding affinity measurements relating to 48 different mouse, human, macaque, and chimpanzee MHC class I alleles. We use this data to establish a set of benchmark predictions with one neural network method and two matrix-based prediction methods extensively utilized in our groups. In general, the neural network outperforms the matrix-based predictions mainly due to its ability to generalize even on a small amount of data. We also retrieved predictions from tools publicly available on the internet. While differences in the data used to generate these predictions hamper direct comparisons, we do conclude that tools based on combinatorial peptide libraries perform remarkably well. The transparent prediction evaluation on this dataset provides tool developers with a benchmark for comparison of newly developed prediction methods. In addition, to generate and evaluate our own prediction methods, we have established an easily extensible web-based prediction framework that allows automated side-by-side comparisons of prediction methods implemented by experts. This is an advance over the current practice of tool developers having to generate reference predictions themselves, which can lead to underestimating the performance of prediction methods they are not as familiar with as their own. The overall goal of this effort is to provide a transparent prediction evaluation allowing bioinformaticians to identify promising features of prediction methods and providing guidance to immunologists regarding the reliability of prediction tools.

Collagen-binding VEGF mimetic peptide: Structure, matrix interaction, and endothelial cell activation

NASA Astrophysics Data System (ADS)

Chan, Tania R.

Long term survival of artificial tissue constructs depends greatly on proper vascularization. In nature, differentiation of endothelial cells and formation of vasculature are directed by dynamic spatio-temporal cues in the extracellular matrix that are difficult to reproduce in vitro. In this dissertation, we present a novel bifunctional peptide that mimics matrix-bound vascular endothelial growth factor (VEGF), which can be used to encode spatially controlled angiogenic signals in collagen-based scaffolds. The peptide, QKCMP, contains a collagen mimetic domain (CMP) that binds to type I collagen by a unique triple helix hybridization mechanism and a VEGF mimetic domain (QK) with pro-angiogenic activity. We demonstrate QKCMP's ability to hybridize with native and heat denatured collagens through a series of binding studies on collagen and gelatin substrates. Circular dichroism experiments show that the peptide retains the triple helical structure vital for collagen binding, and surface plasmon resonance study confirms the molecular interaction between the peptide and collagen strands. Cell culture studies demonstrate QKCMP's ability to induce endothelial cell morphogenesis and network formation as a matrix-bound factor in 2D and 3D collagen scaffolds. We also show that the peptide can be used to spatially modify collagen-based substrates to promote localized endothelial cell activation and network formation. To probe the biological events that govern these angiogenic cellular responses, we investigated the cell signaling pathways activated by collagen-bound QKCMP and determined short and long-term endothelial cell response profiles for p38, ERK1/2, and Akt signal transduction cascades. Finally, we present our efforts to translate the peptide's in vitro bioactivity to an in vivo burn injury animal model. When implanted at the wound site, QKCMP functionalized biodegradable hydrogels induce enhanced neovascularization in the granulation tissue. The results show QKCMP

Widening and diversifying the proteome capture by combinatorial peptide ligand libraries via Alcian Blue dye binding.

PubMed

Candiano, Giovanni; Santucci, Laura; Petretto, Andrea; Lavarello, Chiara; Inglese, Elvira; Bruschi, Maurizio; Ghiggeri, Gian Marco; Boschetti, Egisto; Righetti, Pier Giorgio

2015-01-01

Combinatorial peptide ligand libraries (CPLLs) tend to bind complex molecules such as dyes due to their aromatic, heterocyclic, hydrophobic, and ionic nature that may affect the protein capture specificity. In this experimental work Alcian Blue 8GX, a positively charged phthalocyanine dye well-known to bind to glycoproteins and to glucosaminoglycans, was adsorbed on a chemically modified CPLL solid phase, and the behavior of the resulting conjugate was then investigated. The control and dye-adsorbed beads were used to harvest the human urinary proteome at physiological pH, this resulting in a grand total of 1151 gene products identified after the capture. Although the Alcian Blue-modified CPLL incremented the total protein capture by 115 species, it particularly enriched some families among the harvested proteins, such as glycoproteins and nucleotide-binding proteins. This study teaches that it is possible, via the two combined harvest mechanisms, to drive the CPLL capture toward the enrichment of specific protein categories.

A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

PubMed

Wall, Jonathan S; Williams, Angela; Richey, Tina; Stuckey, Alan; Huang, Ying; Wooliver, Craig; Macy, Sallie; Heidel, Eric; Gupta, Neil; Lee, Angela; Rader, Brianna; Martin, Emily B; Kennel, Stephen J

2013-01-01

Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

Isolation and characterisation of sericin antifreeze peptides and molecular dynamics modelling of their ice-binding interaction.

PubMed

Wu, Jinhong; Rong, Yuzhi; Wang, Zhengwu; Zhou, Yanfu; Wang, Shaoyun; Zhao, Bo

2015-05-01

This study aimed to isolate and characterise a novel sericin antifreeze peptide and investigate its ice-binding molecular mechanism. The thermal hysteresis activity of ice-binding sericin peptides (I-SP) was measured and their activity reached as high as 0.94 °C. A P4 fraction, with high hypothermia protective activity and inhibition activity of ice recrystallisation, was obtained from I-SP, and a purified sericin peptide, named SM-AFP, with the sequence of TTSPTNVSTT and a molecular weight of 1009.50 Da was then isolated from the P4 fraction. Treatment of Lactobacillus delbrueckii Subsp. bulgaricus LB340 LYO with 100 μg/ml synthetic SM-AFP led to 1.4-fold increased survival (p < 0.05). Finally, an SM-AFP/ice binding model was constructed and results of molecular dynamics simulation suggested that the binding of SM-AFP with ice and prevention of ice crystal growth could be attributed to hydrogen bond formation, hydrophobic interaction and non-bond interactions. Sericin peptides could be developed into beneficial cryoprotectants and used in frozen food processing. Copyright © 2014 Elsevier Ltd. All rights reserved.

Unconventional binding sites and receptors for VIP and related peptides PACAP and PHI/PHM: an update.

PubMed

Muller, Jean-Marc; Debaigt, Colin; Goursaud, Stéphanie; Montoni, Alicia; Pineau, Nicolas; Meunier, Annie-Claire; Janet, Thierry

2007-09-01

The 28-amino-acid neuropeptide VIP and related peptides PACAP and PHI/PHM modulate virtually all of the vital functions in the body. These peptides are also commonly recognized as major regulators of cell growth and differentiation. Through their trophic and cytoprotective functions, they appear to play major roles in embryonic development, neurogenesis and the progression of a number of cancer types. These peptides bind to three well-characterized subtypes of G-protein coupled receptors: VPAC1 and VPAC2 share a common high affinity in the nanomolar range for VIP and PACAP; a third receptor type, PAC1, has been characterized for its high affinity for PACAP but its low affinity for VIP. Complex effects and pharmacological behaviors of these peptides suggest that multiple subtypes of binding sites may cooperate to mediate their function in target cells and tissues. In this complex response, some of these binding sites correspond to the definition of the conventional receptors cited above, while others display unexpected pharmacological and functional properties. Here we present potential clues that may lead investigators to further characterize the molecular nature and functions of these atypical binding species.

A Peptide Filtering Relation Quantifies MHC Class I Peptide Optimization

PubMed Central

Goldstein, Leonard D.; Howarth, Mark; Cardelli, Luca; Emmott, Stephen; Elliott, Tim; Werner, Joern M.

2011-01-01

Major Histocompatibility Complex (MHC) class I molecules enable cytotoxic T lymphocytes to destroy virus-infected or cancerous cells, thereby preventing disease progression. MHC class I molecules provide a snapshot of the contents of a cell by binding to protein fragments arising from intracellular protein turnover and presenting these fragments at the cell surface. Competing fragments (peptides) are selected for cell-surface presentation on the basis of their ability to form a stable complex with MHC class I, by a process known as peptide optimization. A better understanding of the optimization process is important for our understanding of immunodominance, the predominance of some T lymphocyte specificities over others, which can determine the efficacy of an immune response, the danger of immune evasion, and the success of vaccination strategies. In this paper we present a dynamical systems model of peptide optimization by MHC class I. We incorporate the chaperone molecule tapasin, which has been shown to enhance peptide optimization to different extents for different MHC class I alleles. Using a combination of published and novel experimental data to parameterize the model, we arrive at a relation of peptide filtering, which quantifies peptide optimization as a function of peptide supply and peptide unbinding rates. From this relation, we find that tapasin enhances peptide unbinding to improve peptide optimization without significantly delaying the transit of MHC to the cell surface, and differences in peptide optimization across MHC class I alleles can be explained by allele-specific differences in peptide binding. Importantly, our filtering relation may be used to dynamically predict the cell surface abundance of any number of competing peptides by MHC class I alleles, providing a quantitative basis to investigate viral infection or disease at the cellular level. We exemplify this by simulating optimization of the distribution of peptides derived from Human

Identification of the bioactive and consensus peptide motif from Momordica charantia insulin receptor-binding protein.

PubMed

Lo, Hsin-Yi; Li, Chia-Cheng; Ho, Tin-Yun; Hsiang, Chien-Yun

2016-08-01

Many food bioactive peptides with diverse functions have been discovered by studying plant proteins. We have previously identified a 68-residue insulin receptor (IR)-binding protein (mcIRBP) from Momordica charantia that exhibits hypoglycemic effects in mice via interaction with IR. By in vitro digestion, we found that mcIRBP-19, spanning residues 50-68 of mcIRBP, enhanced the binding of insulin to IR, stimulated the phosphorylation of PDK1 and Akt, induced the expression of glucose transporter 4, and stimulated both the uptake of glucose in cells and the clearance of glucose in diabetic mice. Furthermore, mcIRBP-19 homologs were present in various plants and shared similar β-hairpin structures and IR kinase-activating abilities to mcIRBP-19. In conclusion, our findings suggested that mcIRBP-19 is a blood glucose-lowering bioactive peptide that exhibits IR-binding potentials. Moreover, we newly identified novel IR-binding bioactive peptides in various plants which belonged to different taxonomic families. Copyright © 2016 Elsevier Ltd. All rights reserved.

Towards development of aptamers that specifically bind to lactate dehydrogenase of Plasmodium falciparum through epitopic targeting.

PubMed

Frith, Kelly-Anne; Fogel, Ronen; Goldring, J P Dean; Krause, Robert G E; Khati, Makobetsa; Hoppe, Heinrich; Cromhout, Mary E; Jiwaji, Meesbah; Limson, Janice L

2018-05-03

Early detection is crucial for the effective treatment of malaria, particularly in those cases infected with Plasmodium falciparum. There is a need for diagnostic devices with the capacity to distinguish P. falciparum from other strains of malaria. Here, aptamers generated against targeted species-specific epitopes of P. falciparum lactate dehydrogenase (rPfLDH) are described. Two classes of aptamers bearing high binding affinity and specificity for recombinant P. falciparum lactate dehydrogenase (rPfLDH) and P. falciparum-specific lactate dehydrogenase epitopic oligopeptide (LDHp) were separately generated. Structurally-relevant moieties with particular consensus sequences (GGTAG and GGCG) were found in aptamers reported here and previously published, confirming their importance in recognition of the target, while novel moieties particular to this work (ATTAT and poly-A stretches) were identified. Aptamers with diagnostically-supportive functions were synthesized, prime examples of which are the aptamers designated as LDHp 1, LDHp 11 and rLDH 4 and rLDH 15 in work presented herein. Of the sampled aptamers raised against the recombinant protein, rLDH 4 showed the highest binding to the target rPfLDH in the ELONA assay, with both rLDH 4 and rLDH 15 indicating an ability to discriminate between rPfLDH and rPvLDH. LDHp 11 was generated against a peptide selected as a unique P. falciparum LDH peptide. The aptamer, LDHp 11, like antibodies against the same peptide, only detected rPfLDH and discriminated between rPfLDH and rPvLDH. This was supported by affinity binding experiments where only aptamers generated against a unique species-specific epitope showed an ability to preferentially bind to rPfLDH relative to rPvLDH rather than those generated against the whole recombinant protein. In addition, rLDH 4 and LDHp 11 demonstrated in situ binding to P. falciparum cells during confocal microscopy. The utilization and application of LDHp 11, an aptamer generated against a

Bioelectronic Nose Using Odorant Binding Protein-Derived Peptide and Carbon Nanotube Field-Effect Transistor for the Assessment of Salmonella Contamination in Food.

PubMed

Son, Manki; Kim, Daesan; Kang, Jinkyung; Lim, Jong Hyun; Lee, Seung Hwan; Ko, Hwi Jin; Hong, Seunghun; Park, Tai Hyun

2016-12-06

Salmonella infection is the one of the major causes of food borne illnesses including fever, abdominal pain, diarrhea, and nausea. Thus, early detection of Salmonella contamination is important for our healthy life. Conventional detection methods for the food contamination have limitations in sensitivity and rapidity; thus, the early detection has been difficult. Herein, we developed a bioelectronic nose using a carbon nanotube (CNT) field-effect transistor (FET) functionalized with Drosophila odorant binding protein (OBP)-derived peptide for easy and rapid detection of Salmonella contamination in ham. 3-Methyl-1-butanol is known as a specific volatile organic compound, generated from the ham contaminated with Salmonella. We designed and synthesized the peptide based on the sequence of the Drosophila OBP, LUSH, which specifically binds to alcohols. The C-terminus of the synthetic peptide was modified with three phenylalanine residues and directly immobilized onto CNT channels using the π-π interaction. The p-type properties of FET were clearly maintained after the functionalization using the peptide. The biosensor detected 1 fM of 3-methyl-1-butanol with high selectivity and successfully assessed Salmonella contamination in ham. These results indicate that the bioelectronic nose can be used for the rapid detection of Salmonella contamination in food.

Distinct mechanisms of a phosphotyrosyl peptide binding to two SH2 domains.

PubMed

Pang, Xiaodong; Zhou, Huan-Xiang

2014-05-01

Protein phosphorylation is very common post-translational modification, catalyzed by kinases, for signaling and regulation. Phosphotyrosines frequently target SH2 domains. The spleen tyrosine kinase (Syk) is critical for tyrosine phosphorylation of multiple proteins and for regulation of important pathways. Phosphorylation of both Y342 and Y346 in Syk linker B is required for optimal signaling. The SH2 domains of Vav1 and PLC-γ both bind this doubly phosphorylated motif. Here we used a recently developed method to calculate the effects of Y342 and Y346 phosphorylation on the rate constants of a peptide from Syk linker B binding to the SH2 domains of Vav1 and PLC-γ. The predicted effects agree well with experimental observations. Moreover, we found that the same doubly phosphorylated peptide binds the two SH2 domains via distinct mechanisms, with apparent rigid docking for Vav1 SH2 and dock-and-coalesce for PLC-γ SH2.

Design of specific peptide inhibitors of phospholipase A2: structure of a complex formed between Russell's viper phospholipase A2 and a designed peptide Leu-Ala-Ile-Tyr-Ser (LAIYS).

PubMed

Chandra, Vikas; Jasti, Jayasankar; Kaur, Punit; Dey, Sharmistha; Srinivasan, A; Betzel, Ch; Singh, T P

2002-10-01

Phospholipase A(2) (EC 3.1.1.4) is a key enzyme of the cascade mechanism involved in the production of proinflammatory compounds known as eicosanoids. The binding of phospholipase A(2) to membrane surfaces and the hydrolysis of phospholipids are thought to involve the formation of a hydrophobic channel into which a single substrate molecule diffuses before cleavage. In order to regulate the production of proinflammatory compounds, a specific peptide inhibitor of PLA(2), Leu-Ala-Ile-Tyr-Ser, has been designed. Phospholipase A(2) from Daboia russelli pulchella (DPLA(2)) and peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) have been co-crystallized. The structure of the complex has been determined and refined to 2.0 A resolution. The structure contains two crystallographically independent molecules of DPLA(2), with one molecule of peptide specifically bound to one of them. The overall conformations of the two molecules are essentially similar except in three regions; namely, the calcium-binding loop including Trp31 (residues 25-34), the beta-wing consisting of two antiparallel beta-strands (residues 74-85) and the C-terminal region (residues 119-133). Of these, the most striking difference pertains to the orientation of Trp31 in the two molecules. The conformation of Trp31 in molecule A was suitable to allow the binding of peptide LAIYS, while that in molecule B prevented the entry of the ligand into the hydrophobic channel. The structure of the complex clearly showed that the OH group of Tyr of the inhibitor formed hydrogen bonds with both His48 N(delta1) and Asp49 O(delta1), while O(gamma)H of Ser was involved in a hydrogen bond with Trp31. Other peptide backbone atoms interact with protein through water molecules, while Leu, Ala and Ile form strong hydrophobic interactions with the residues of the hydrophobic channel.

Binding Modes of Thioflavin T Molecules to Prion Peptide Assemblies Identified by Using Scanning Tunneling Microscopy

PubMed Central

2011-01-01

The widely used method to monitor the aggregation process of amyloid peptide is thioflavin T (ThT) assay, while the detailed molecular mechanism is still not clear. In this work, we report here the direct identification of the binding modes of ThT molecules with the prion peptide GNNQQNY by using scanning tunneling microscopy (STM). The assembly structures of GNNQQNY were first observed by STM on a graphite surface, and the introduction of ThT molecules to the surface facilitated the STM observations of the adsorption conformations of ThT with peptide strands. ThT molecules are apt to adsorb on the peptide assembly with β-sheet structure and oriented parallel with the peptide strands adopting four different binding modes. This effort could benefit the understanding of the mechanisms of the interactions between labeling species or inhibitory ligands and amyloid peptides, which is keenly needed for developing diagnostic and therapeutic approaches. PMID:22778872

The Baculovirus-Expressed Binding Region of Plasmodium falciparum EBA-140 Ligand and Its Glycophorin C Binding Specificity

PubMed Central

Rydzak, Joanna; Kaczmarek, Radoslaw; Czerwinski, Marcin; Lukasiewicz, Jolanta; Tyborowska, Jolanta; Szewczyk, Boguslaw; Jaskiewicz, Ewa

2015-01-01

The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during invasion. In this report we describe, for the first time, the glycophorin C specificity of the recombinant, baculovirus-expressed binding region (Region II) of P. falciparum EBA-140 ligand. It was found that the recombinant EBA-140 Region II binds to the endogenous and recombinant glycophorin C, but does not bind to Gerbich-type glycophorin C, neither normal nor recombinant, which lacks amino acid residues 36–63 of its polypeptide chain. Our results emphasize the crucial role of this glycophorin C region in EBA-140 ligand binding. Moreover, the EBA-140 Region II did not bind either to glycophorin D, the truncated form of glycophorin C lacking the N-glycan or to desialylated GPC. These results draw attention to the role of glycophorin C glycans in EBA-140 binding. The full identification of the EBA-140 binding site on glycophorin C molecule, consisting most likely of its glycans and peptide backbone, may help to design therapeutics or vaccines that target the erythrocyte binding merozoite ligands. PMID:25588042

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

NASA Technical Reports Server (NTRS)

Safadi, F.; Reddy, V. S.; Reddy, A. S.

2000-01-01

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

The electrostatic surface of MDM2 modulates the specificity of its interaction with phosphorylated and unphosphorylated p53 peptides.

PubMed

Brown, Christopher John; Srinivasan, Deepa; Jun, Lee Hui; Coomber, David; Verma, Chandra S; Lane, David P

2008-03-01

Florescence anisotropy measurements using FAM-labelled p53 peptides showed that the binding of the peptides to MDM2 was dependant upon the phosphorylation of p53 at Thr18 and that this binding was modulated by the electrostatic properties of MDM2. In agreement with computational predictions, the binding to phosphorylated p53 peptide, in comparison to the unphosphorylated p53 peptide, was enhanced upon mutation of 3 key residues on the MDM2 surface.

Organic crystal-binding peptides: morphology control and one-pot formation of protein-displaying organic crystals

NASA Astrophysics Data System (ADS)

Niide, Teppei; Ozawa, Kyohei; Nakazawa, Hikaru; Oliveira, Daniel; Kasai, Hitoshi; Onodera, Mari; Asano, Ryutaro; Kumagai, Izumi; Umetsu, Mitsuo

2015-11-01

Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals.Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar

Regulator of differentiation 1 (ROD1) binds to the amphipathic C-terminal peptide of thrombospondin-4 and is involved in its mitogenic activity.

PubMed

Sadvakassova, Gulzhakhan; Dobocan, Monica C; Difalco, Marcos R; Congote, Luis F

2009-09-01

The matrix protein thrombospondin-4 has an acidic amphipathic C-terminal peptide (C21) which stimulates erythroid cell proliferation. Here we show that C21 stimulates red cell formation in anemic mice in vivo. In vitro experiments indicated that the peptide-mediated increase of erythroid colony formation in cultures of human CD34+ hematopoietic progenitor cells was possible only under continuous presence of erythropoietin. In the absence of this cytokine, C21 stimulated exclusively myeloid colony formation. Therefore, the peptide is not a specific erythroid differentiation factor. In fact, it is mitogenic in non-erythroid cells, such as skin fibroblasts and kidney epithelial cells. In erythroleukemic TF-1 cells, it actually decreased the production of the erythroid differentiation marker glycophorin A. C21-affinity chromatography revealed regulator of differentiation 1 (ROD1) as a major C21-binding protein. ROD1 is the hematopoietic cell paralog of polypyrimidine tract binding proteins (PTBs), RNA splice regulators which regulate differentiation by repressing tissue-specific exons. ROD1 binding to C21 was strongly inhibited by synthetic RNAs in the order poly A > poly U > poly G = poly C and was weakly inhibited by a synthetic phosphorylated peptide mimicking the C-terminal domain of RNA polymerase II. Cellular overexpression or knockdown experiments of ROD1 suggest a role for this protein in the mitogenic activity of C21. Since the nuclear proteins ROD1 and PTBs regulate differentiation at a posttranscriptional level and there is a fast nuclear uptake of C21, we put forward the idea that the peptide is internalized, goes to the nucleus and maintains cells in a proliferative state by supporting ROD1-mediated inhibition of differentiation.

Structural Analysis of Semi-specific Oligosaccharide Recognition by a Cellulose-binding Protein of Thermotoga maritima Reveals Adaptations for Functional Diversification of the Oligopeptide Periplasmic Binding Protein Fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cuneo, Matthew J.; Beese, Lorena S.; Hellinga, Homme W.

Periplasmic binding proteins (PBPs) constitute a protein superfamily that binds a wide variety of ligands. In prokaryotes, PBPs function as receptors for ATP-binding cassette or tripartite ATP-independent transporters and chemotaxis systems. In many instances, PBPs bind their cognate ligands with exquisite specificity, distinguishing, for example, between sugar epimers or structurally similar anions. By contrast, oligopeptide-binding proteins bind their ligands through interactions with the peptide backbone but do not distinguish between different side chains. The extremophile Thermotoga maritima possesses a remarkable array of carbohydrate-processing metabolic systems, including the hydrolysis of cellulosic polymers. Here, we present the crystal structure of a T.more » maritima cellobiose-binding protein (tm0031) that is homologous to oligopeptide-binding proteins. T. maritima cellobiose-binding protein binds a variety of lengths of {beta}(1 {yields} 4)-linked glucose oligomers, ranging from two rings (cellobiose) to five (cellopentaose). The structure reveals that binding is semi-specific. The disaccharide at the nonreducing end binds specifically; the other rings are located in a large solvent-filled groove, where the reducing end makes several contacts with the protein, thereby imposing an upper limit of the oligosaccharides that are recognized. Semi-specific recognition, in which a molecular class rather than individual species is selected, provides an efficient solution for the uptake of complex mixtures.« less

A Cyclic Peptidic Serine Protease Inhibitor: Increasing Affinity by Increasing Peptide Flexibility

PubMed Central

Jiang, Longguang; Paaske, Berit; Kromann-Hansen, Tobias; Jensen, Jan K.; Sørensen, Hans Peter; Liu, Zhuo; Nielsen, Jakob T.; Christensen, Anni; Hosseini, Masood; Sørensen, Kasper K.; Nielsen, Niels Christian; Jensen, Knud J.; Huang, Mingdong; Andreasen, Peter A.

2014-01-01

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden. PMID:25545505

Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

PubMed Central

David, Marion; Lécorché, Pascaline; Masse, Maxime; Faucon, Aude; Abouzid, Karima; Gaudin, Nicolas; Varini, Karine; Gassiot, Fanny; Ferracci, Géraldine; Jacquot, Guillaume; Vlieghe, Patrick

2018-01-01

Insufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH411 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or ldlr-/- mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells -including pathological cells—or organs that express the LDLR. PMID:29485998

Cationic Peptides and Peptidomimetics Bind Glycosaminoglycans as Potential Sema3A Pathway Inhibitors

PubMed Central

Corredor, Miriam; Bonet, Roman; Moure, Alejandra; Domingo, Cecilia; Bujons, Jordi; Alfonso, Ignacio; Pérez, Yolanda; Messeguer, Àngel

2016-01-01

Semaphorin3A (Sema3A) is a vertebrate-secreted protein that was initially characterized as a repulsive-guidance cue. Semaphorins have crucial roles in several diseases; therefore, the development of Sema3A inhibitors is of therapeutic interest. Sema3A interacts with glycosaminoglycans (GAGs), presumably through its C-terminal basic region. We used different biophysical techniques (i.e., NMR, surface plasmon resonance, isothermal titration calorimetry, fluorescence, and UV-visible spectroscopy) to characterize the binding of two Sema3A C-terminus-derived basic peptides (FS2 and NFS3) to heparin and chondroitin sulfate A. We found that these peptides bind to both GAGs with affinities in the low-micromolar range. On the other hand, a peptoid named SICHI (semaphorin-induced chemorepulsion inhibitor), which is positively charged at physiological pH, was first identified by our group as being able to block Sema3A chemorepulsion and growth-cone collapse in axons at the extracellular level. To elucidate the direct target for the reported SICHI inhibitory effect in the Sema3A signaling pathway, we looked first to the protein-protein interaction between secreted Sema3A and the Nrp1 receptor. However, our results show that SICHI does not bind directly to the Sema3A sema domain or to Nrp1 extracellular domains. We evaluated a new, to our knowledge, hypothesis, according to which SICHI binds to GAGs, thereby perturbing the Sema3A-GAG interaction. By using the above-mentioned techniques, we observed that SICHI binds to GAGs and competes with Sema3A C-terminus-derived basic peptides for binding to GAGs. These data support the ability of SICHI to block the biologically relevant interaction between Sema3A and GAGs, thus revealing SICHI as a new, to our knowledge, class of inhibitors that target the GAG-protein interaction. PMID:27028639

Pharmacological lineage analysis revealed the binding affinity of broad-spectrum substance P antagonists to receptors for gonadotropin-releasing peptide.

PubMed

Arai, Kazune; Kashiwazaki, Aki; Fujiwara, Yoko; Tsuchiya, Hiroyoshi; Sakai, Nobuya; Shibata, Katsushi; Koshimizu, Taka-aki

2015-02-15

A group of synthetic substance P (SP) antagonists, such as [Arg(6),D-Trp(7,9),N(Me)Phe(8)]-substance P(6-11) and [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]-substance P, bind to a range of distinct G-protein-coupled receptor (GPCR) family members, including V1a vasopressin receptors, and they competitively inhibit agonist binding. This extended accessibility enabled us to identify a GPCR subset with a partially conserved binding site structure. By combining pharmacological data and amino acid sequence homology matrices, a pharmacological lineage of GPCRs that are sensitive to these two SP antagonists was constructed. We found that sensitivity to the SP antagonists was not limited to the Gq-protein-coupled V1a and V1b receptors; Gs-coupled V2 receptors and oxytocin receptors, which couple with both Gq and Gi, also demonstrated sensitivity. Unexpectedly, a dendrogram based on the amino acid sequences of 222 known GPCRs showed that a group of receptors sensitive to the SP antagonists are located in close proximity to vasopressin/oxytocin receptors. Gonadotropin-releasing peptide receptors, located near the vasopressin receptors in the dendrogram, were also sensitive to the SP analogs, whereas α1B adrenergic receptors, located more distantly from the vasopressin receptors, were not sensitive. Our finding suggests that pharmacological lineage analysis is useful in selecting subsets of candidate receptors that contain a conserved binding site for a ligand with broad-spectrum binding abilities. The knowledge that the binding site of the two broad-spectrum SP analogs partially overlaps with that of distinct peptide agonists is valuable for understanding the specificity/broadness of peptide ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

Lactobacillus buchneri S-layer as carrier for an Ara h 2-derived peptide for peanut allergen-specific immunotherapy.

PubMed

Anzengruber, Julia; Bublin, Merima; Bönisch, Eva; Janesch, Bettina; Tscheppe, Angelika; Braun, Matthias L; Varga, Eva-Maria; Hafner, Christine; Breiteneder, Heimo; Schäffer, Christina

2017-05-01

Peanut allergy is an IgE-mediated severe hypersensitivity disorder. The lack of a treatment of this potentially fatal allergy has led to intensive research on vaccine development. Here, we describe the design and initial characterization of a carrier-bound peptide derived from the most potent peanut allergen, Ara h 2, as a candidate vaccine. Based on the adjuvant capability of bacterial surface (S-) layers, a fusion protein of the S-layer protein SlpB from Lactobacillus buchneri CD034 and the Ara h 2-derived peptide AH3a42 was produced. This peptide comprised immunodominant B-cell epitopes as well as one T cell epitope. The fusion protein SlpB-AH3a42 was expressed in E. coli, purified, and tested for its IgE binding capacity as well as for its ability to activate sensitized rat basophil leukemia (RBL) cells. The capacity of Ara h 2-specific IgG rabbit-antibodies raised against SlpB-AH3a42 or Ara h 2 to inhibit IgE-binding was determined by ELISA inhibition assays using sera of peanut allergic patients sensitized to Ara h 2. IgE specific to the SlpB-AH3a42 fusion protein was detected in 69% (25 of 36) of the sera. Despite the recognition by IgE, the SlpB-AH3a42 fusion protein was unable to induce β-hexosaminidase release from sensitized RBL cells at concentrations up to 100ng per ml. The inhibition of IgE-binding to the natural allergen observed after pre-incubation of the 20 sera with rabbit anti-SlpB-AH3a42 IgG was more than 30% for four sera, more than 20% for eight sera, and below 10% for eight sera. In comparison, anti-Ara h 2 rabbit IgG antibodies inhibited binding to Ara h 2 by 48% ±13.5%. Our data provide evidence for the feasibility of this novel approach towards the development of a peanut allergen peptide-based carrier-bound vaccine. Our experiments further indicate that more than one allergen-peptide will be needed to induce a broader protection of patients allergic to Ara h 2. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights

Characterization of the interaction forces in a drug carrier complex of doxorubicin with a drug-binding peptide.

PubMed

Gocheva, Gergana; Ilieva, Nina; Peneva, Kalina; Ivanova, Anela

2018-04-01

Polypeptide-based materials are used as building blocks for drug delivery systems aimed at toxicity decrease in chemotherapeutics. A molecular-level approach is adopted for investigating the non-covalent interactions between doxorubicin and a recently synthesized drug-binging peptide as a key part of a system for delivery to neoplastic cells. Molecular dynamics simulations in aqueous solution at room and body temperature are applied to investigate the structure and the binding modes within the drug-peptide complex. The tryptophans are outlined as the main chemotherapeutic adsorption sites, and the importance of their placement in the peptide sequence is highlighted. The drug-peptide binging energy is evaluated by density functional theory calculations. Principal component analysis reveals comparable importance of several types of interaction for the binding strength. π-Stacking is dominant, but other factors are also significant: intercalation, peptide backbone stacking, electrostatics, dispersion, and solvation. Intra- and intermolecular H-bonding also stabilizes the complexes. The influence of solvent molecules on the binding energy is mild. The obtained data characterize the drug-to-peptide attachment as a mainly attractive collective process with interactions spanning a broad range of values. These results explain with atomistic detail the experimentally registered doxorubicin-binging ability of the peptide and outline the complex as a prospective carrying unit that can be employed in design of drug delivery systems. © 2017 John Wiley & Sons A/S.

Simultaneous alignment and clustering of peptide data using a Gibbs sampling approach.

PubMed

Andreatta, Massimo; Lund, Ole; Nielsen, Morten

2013-01-01

Proteins recognizing short peptide fragments play a central role in cellular signaling. As a result of high-throughput technologies, peptide-binding protein specificities can be studied using large peptide libraries at dramatically lower cost and time. Interpretation of such large peptide datasets, however, is a complex task, especially when the data contain multiple receptor binding motifs, and/or the motifs are found at different locations within distinct peptides. The algorithm presented in this article, based on Gibbs sampling, identifies multiple specificities in peptide data by performing two essential tasks simultaneously: alignment and clustering of peptide data. We apply the method to de-convolute binding motifs in a panel of peptide datasets with different degrees of complexity spanning from the simplest case of pre-aligned fixed-length peptides to cases of unaligned peptide datasets of variable length. Example applications described in this article include mixtures of binders to different MHC class I and class II alleles, distinct classes of ligands for SH3 domains and sub-specificities of the HLA-A*02:01 molecule. The Gibbs clustering method is available online as a web server at http://www.cbs.dtu.dk/services/GibbsCluster.

Revisiting the arthritogenic peptide theory: quantitative not qualitative changes in the peptide repertoire of HLA-B27 allotypes.

PubMed

Schittenhelm, Ralf B; Sian, Terry C C Lim Kam; Wilmann, Pascal G; Dudek, Nadine L; Purcell, Anthony W

2015-03-01

The association of HLA-B27 with spondyloarthropathy is one of the strongest documented for any autoimmune disease. A common hypothesis for this association is the arthritogenic peptide concept. This dictates that differences in the peptide binding preferences of disease-associated and non-disease-associated HLA-B27 allotypes underlie the presentation of bacterial and self-peptides, leading to cross-reactive T cell immunity and subsequent autoimmune attack of affected tissues. The aim of this study was to analyze and compare self-peptides from 8 HLA-B27 allotypes, to increase existing data sets of HLA-B27 ligands, to refine and compare their consensus-binding motifs, and to reveal similarities and differences in the peptide repertoire of the HLA-B27 subtypes. Qualitative differences in the peptides bound to the 8 most frequent HLA-B27 subtypes were determined by tandem mass spectrometry, and quantitative changes in allelic binding specificities were determined by highly sensitive and targeted multiple reaction monitoring mass spectrometry. We identified >7,500 major histocompatibility complex class I peptides derived from the 8 most common HLA-B27 allotypes (HLA-B*27:02 to HLA-B*27:09). We describe individual binding motifs for these alleles for the 9-12-mer ligands. The peptide repertoires of these closely related alleles showed significant overlap. Allelic polymorphisms resulting in changes in the amino acid composition of the antigen-binding cleft manifested largely as quantitative changes in the peptide cargo of these molecules. Absolute binding preferences of HLA-B27 allotypes do not explain disease association. The arthritogenic peptide theory needs to be reassessed in terms of quantitative changes in self-peptide presentation, T cell selection, and altered conformation of bound peptides. Copyright © 2015 by the American College of Rheumatology.

MUB40 Binds to Lactoferrin and Stands as a Specific Neutrophil Marker.

PubMed

Anderson, Mark C; Chaze, Thibault; Coïc, Yves-Marie; Injarabian, Louise; Jonsson, Friederike; Lombion, Naelle; Selimoglu-Buet, Dorothée; Souphron, Judith; Ridley, Caroline; Vonaesch, Pascale; Baron, Bruno; Arena, Ellen T; Tinevez, Jean-Yves; Nigro, Giulia; Nothelfer, Katharina; Solary, Eric; Lapierre, Valérie; Lazure, Thierry; Matondo, Mariette; Thornton, David; Sansonetti, Philippe J; Baleux, Françoise; Marteyn, Benoit S

2018-04-19

Neutrophils represent the most abundant immune cells recruited to inflamed tissues. A lack of dedicated tools has hampered their detection and study. We show that a synthesized peptide, MUB 40 , binds to lactoferrin, the most abundant protein stored in neutrophil-specific and tertiary granules. Lactoferrin is specifically produced by neutrophils among other leukocytes, making MUB 40 a specific neutrophil marker. Naive mammalian neutrophils (human, guinea pig, mouse, rabbit) were labeled by fluorescent MUB 40 conjugates (-Cy5, Dylight405). A peptidase-resistant retro-inverso MUB 40 (RI-MUB 40 ) was synthesized and its lactoferrin-binding property validated. Neutrophil lactoferrin secretion during in vitro Shigella infection was assessed with RI-MUB 40 -Cy5 using live cell microscopy. Systemically administered RI-MUB 40 -Cy5 accumulated at sites of inflammation in a mouse arthritis inflammation model in vivo and showed usefulness as a potential tool for inflammation detection using non-invasive imaging. Improving neutrophil detection with the universal and specific MUB 40 marker will aid the study of broad ranges of inflammatory diseases. Copyright © 2018 Elsevier Ltd. All rights reserved.

A 31-residue peptide induces aggregation of tau’s microtubule-binding region in cells

PubMed Central

Stöhr, Jan; Wu, Haifan; Nick, Mimi; Wu, Yibing; Bhate, Manasi; Condello, Carlo; Johnson, Noah; Rodgers, Jeffrey; Lemmin, Thomas; Achyraya, Srabasti; Becker, Julia; Robinson, Kathleen; Kelly, Mark J.S.; Gai, Feng; Stubbs, Gerald; Prusiner, Stanley B.; DeGrado, William F.

2018-01-01

The self-propagation of misfolded conformations of tau underlies neurodegenerative diseases, including Alzheimer’s disease. There is considerable interest in discovering the minimal sequence and active conformational nucleus that defines this self-propagating event. The microtubule-binding region, spanning residues 244-372, reproduces much of the aggregation behavior of tau in cells and animal models. Further dissection of the amyloid-forming region to a hexapeptide from the third microtubule-binding repeat resulted in a peptide that rapidly forms fibrils in vitro. We show here that this peptide lacks the ability to seed aggregation of tau244-372 in cells. However, as the hexapeptide is gradually extended to 31 residues, the peptides aggregate more slowly and gain potent activity to induce aggregation of tau244-372 in cells. X-ray fiber diffraction, hydrogen-deuterium exchange and solids NMR studies map the beta-forming region to a 25-residue sequence. Thus, the nucleus for self-propagating aggregation of tau244-372 in cells is packaged in a remarkably small peptide. PMID:28837163

Specific high-affinity binding sites for a synthetic gliadin heptapeptide of human peripheral blood lymphocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Payan, D.G.; Horvath, K.; Graf, L.

1987-03-23

The synthetic peptide containing residues 43-49 of ..cap alpha..-gliadin, the major protein component of gluten, has previously been shown to inhibit the production of lymphokine activities by mononuclear leukocytes. The authors demonstrate using radiolabeled ..cap alpha..-gliadin(43-49) that human peripheral blood lymphocytes express approximately 20,000-25,000 surface receptors for this peptide, with a dissociation constant (K/sub D/) of 20 nM. In addition, binding is inhibited by naloxone and an enkephalin analog, thus confirming the functional correlate which demonstrates inhibition by these agents of ..cap alpha..-gliadin(43-49) functional effects. Furthermore, B-lymphocytes bind specifically a greater amount of (/sup 125/I)..cap alpha..-gliadin(43-49) than T-lymphocytes. The lymphocytemore » ..cap alpha..-gliadin(43-49) receptor may play an important role in mediating the immunological response to ..cap alpha..-gliadin. 16 references, 4 figures.« less

Binding of Disordered Peptides to Kelch: Insights from Enhanced Sampling Simulations.

PubMed

Do, Trang Nhu; Choy, Wing-Yiu; Karttunen, Mikko

2016-01-12

Keap1 protein plays an essential role in regulating cellular oxidative stress response and is a crucial binding hub for multiple proteins, several of which are intrinsically disordered proteins (IDP). Among Kelch's IDP binding partners, NRF2 and PTMA are the two most interesting cases. They share a highly similar binding motif; however, NRF2 binds to Kelch with a binding affinity of approximately 100-fold higher than that of PTMA. In this study, we perform an exhaustive sampling composed of 6 μs well-tempered metadynamics and 2 μs unbiased molecular dynamics (MD) simulations aiming at characterizing the binding mechanisms and structural properties of these two peptides. Our results agree with previous experimental observations that PTMA is remarkably more disordered than NRF2 in both the free and bound states. This explains PTMA's lower binding affinity. Our extensive sampling also provides valuable insights into the vast conformational ensembles of both NRF2 and PTMA, supports the hypothesis of coupled folding-binding, and confirms the essential role of linear motifs in IDP binding.

Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer

NASA Astrophysics Data System (ADS)

McGuire, Michael J.; Gray, Bethany Powell; Li, Shunzi; Cupka, Dorothy; Byers, Lauren Averett; Wu, Lei; Rezaie, Shaghayegh; Liu, Ying-Horng; Pattisapu, Naveen; Issac, James; Oyama, Tsukasa; Diao, Lixia; Heymach, John V.; Xie, Xian-Jin; Minna, John D.; Brown, Kathlynn C.

2014-03-01

Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071-40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands.

Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer

PubMed Central

McGuire, Michael J.; Gray, Bethany Powell; Li, Shunzi; Cupka, Dorothy; Byers, Lauren Averett; Wu, Lei; Rezaie, Shaghayegh; Liu, Ying-Horng; Pattisapu, Naveen; Issac, James; Oyama, Tsukasa; Diao, Lixia; Heymach, John V.; Xie, Xian-Jin; Minna, John D.; Brown, Kathlynn C.

2014-01-01

Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071–40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands. PMID:24670678

Molecular Mechanism of Thioflavin-T Binding to the Surface of [beta]-Rich Peptide Self-Assemblies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biancalana, Matthew; Makabe, Koki; Koide, Akiko

A number of small organic molecules have been developed that bind to amyloid fibrils, a subset of which also inhibit fibrillization. Among these, the benzothiol dye Thioflavin-T (ThT) has been used for decades in the diagnosis of protein-misfolding diseases and in kinetic studies of self-assembly (fibrillization). Despite its importance, efforts to characterize the ThT-binding mechanism at the atomic level have been hampered by the inherent insolubility and heterogeneity of peptide self-assemblies. To overcome these challenges, we have developed a minimalist approach to designing a ThT-binding site in a 'peptide self-assembly mimic' (PSAM) scaffold. PSAMs are engineered water-soluble proteins that mimicmore » a segment of beta-rich peptide self-assembly, and they are amenable to standard biophysical techniques and systematic mutagenesis. The PSAM beta-sheet contains rows of repetitive amino acid patterns running perpendicular to the strands (cross-strand ladders) that represent a ubiquitous structural feature of fibril-like surfaces. We successfully designed a ThT-binding site that recapitulates the hallmarks of ThT-fibril interactions by constructing a cross-strand ladder consisting of contiguous tyrosines. The X-ray crystal structures suggest that ThT interacts with the beta-sheet by docking onto surfaces formed by a single tyrosine ladder, rather than in the space between adjacent ladders. Systematic mutagenesis further demonstrated that tyrosine surfaces across four or more beta-strands formed the minimal binding site for ThT. Our work thus provides structural insights into how this widely used dye recognizes a prominent subset of peptide self-assemblies, and proposes a strategy to elucidate the mechanisms of fibril-ligand interactions.« less

Antigenic peptides containing large PEG loops designed to extend out of the HLA-A2 binding site form stable complexes with class I major histocompatibility complex molecules.

PubMed Central

Bouvier, M; Wiley, D C

1996-01-01

Recognition of peptides bound to class I major histocompatibility complex (MHC) molecules by specific receptors on T cells regulates the development and activity of the cellular immune system. We have designed and synthesized de novo cyclic peptides that incorporate PEG in the ring structure for binding to class I MHC molecules. The large PEG loops are positioned to extend out of the peptide binding site, thus creating steric effects aimed at preventing the recognition of class I MHC complexes by T-cell receptors. Peptides were synthesized and cyclized on polymer support using high molecular weight symmetrical PEG dicarboxylic acids to link the side chains of lysine residues substituted at positions 4 and 8 in the sequence of the HLA-A2-restricted human T-lymphotrophic virus type I Tax peptide. Cyclic peptides promoted the in vitro folding and assembly of HLA-A2 complexes. Thermal denaturation studies using circular dichroism spectroscopy showed that these complexes are as stable as complexes formed with antigenic peptides. Images Fig. 2 Fig. 4 PMID:8643447

Linker length dependent binding of a focal adhesion kinase derived peptide to the Src SH3-SH2 domains.

PubMed

Lindfors, Hanna E; Venkata, Bharat Somireddy; Drijfhout, Jan W; Ubbink, Marcellus

2011-02-18

The interaction between a peptide encompassing the SH3 and SH2 binding motifs of focal adhesion kinase (FAK) and the Src SH3-SH2 domains has been investigated with NMR spectroscopy and calorimetry. The binding to both motifs is anti-cooperative. Reduction of the long linker connecting the motifs does not lead to cooperativity. Short linkers that do not allow simultaneous intramolecular binding of the peptide to both motifs cause peptide-mediated dimerisation, even with a linker of only three amino acids. The role of the SH3 binding motif is discussed in view of the independent nature of the SH interactions. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Transmissible gastroenteritis virus; identification of M protein-binding peptide ligands with antiviral and diagnostic potential

USDA-ARS?s Scientific Manuscript database

The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and ...

Engineering peptide ligase specificity by proteomic identification of ligation sites.

PubMed

Weeks, Amy M; Wells, James A

2018-01-01

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme-substrate specificity limits its utility. We developed an approach for comprehensively characterizing peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins, including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. Our methods provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

Structure-based characterization of the binding of peptide to the human endophilin-1 Src homology 3 domain using position-dependent noncovalent potential analysis.

PubMed

Fu, Chunjiang; Wu, Gang; Lv, Fenglin; Tian, Feifei

2012-05-01

Many protein-protein interactions are mediated by a peptide-recognizing domain, such as WW, PDZ, or SH3. In the present study, we describe a new method called position-dependent noncovalent potential analysis (PDNPA), which can accurately characterize the nonbonding profile between the human endophilin-1 Src homology 3 (hEndo1 SH3) domain and its peptide ligands and quantitatively predict the binding affinity of peptide to hEndo1 SH3. In this procedure, structure models of diverse peptides in complex with the hEndo1 SH3 domain are constructed by molecular dynamics simulation and a virtual mutagenesis protocol. Subsequently, three noncovalent interactions associated with each position of the peptide ligand in the complexed state are analyzed using empirical potential functions, and the resulting potential descriptors are then correlated with the experimentally measured affinity on the basis of 1997 hEndo1 SH3-binding peptides with known activities, using linear partial least squares regression (PLS) and the nonlinear support vector machine (SVM). The results suggest that: (i) the electrostatics appears to be more important than steric properties and hydrophobicity in the formation of the hEndo1 SH3-peptide complex; (ii) P(-4) of the core decapeptide ligand with the sequence pattern P(-6)P(-5)P(-4)P(-3)P(-2)P(-1)P(0)P(1)P(2)P(3) is the most important position in terms of determining both the stability and specificity of the architecture of the complex, and; (iii) nonlinear SVM appears to be more effective than linear PLS for accurately predicting the binding affinity of a peptide ligand to hEndo1 SH3, whereas PLS models are straightforward and easy to interpret as compared to those built by SVM.

BiodMHC: an online server for the prediction of MHC class II-peptide binding affinity.

PubMed

Wang, Lian; Pan, Danling; Hu, Xihao; Xiao, Jinyu; Gao, Yangyang; Zhang, Huifang; Zhang, Yan; Liu, Juan; Zhu, Shanfeng

2009-05-01

Effective identification of major histocompatibility complex (MHC) molecules restricted peptides is a critical step in discovering immune epitopes. Although many online servers have been built to predict class II MHC-peptide binding affinity, they have been trained on different datasets, and thus fail in providing a unified comparison of various methods. In this paper, we present our implementation of seven popular predictive methods, namely SMM-align, ARB, SVR-pairwise, Gibbs sampler, ProPred, LP-top2, and MHCPred, on a single web server named BiodMHC (http://biod.whu.edu.cn/BiodMHC/index.html, the software is available upon request). Using a standard measure of AUC (Area Under the receiver operating characteristic Curves), we compare these methods by means of not only cross validation but also prediction on independent test datasets. We find that SMM-align, ProPred, SVR-pairwise, ARB, and Gibbs sampler are the five best-performing methods. For the binding affinity prediction of class II MHC-peptide, BiodMHC provides a convenient online platform for researchers to obtain binding information simultaneously using various methods.

RNA detection using peptide-inserted Renilla luciferase.

PubMed

Andou, Takashi; Endoh, Tamaki; Mie, Masayasu; Kobatake, Eiry

2009-01-01