Detection of peptides lies at the core of bottom-up proteomics analyses. We examined a Bayesian approach to peptide detection, integrating match-based models (fragments, retention time, isotopic distribution, and precursor mass) and peptide prior probability models under a unified probabilistic framework. To assess the relevance of these models and their various combinations, we employed a complete- and a tail-complete search of a low-precursor-mass synthetic peptide library based on oncogenic KRAS peptides. The fragment match was by far the most informative match-based model, while the retention time match was the only remaining such model with an appreciable impact--increasing correct detections by around 8 %. A peptide prior probability model built from a reference proteome greatly improved the detection over a uniform prior, essentially transforming de novo sequencing into a reference-guided search. The knowledge of a correct sequence tag in advance to peptide-spectrum matching had only a moderate impact on peptide detection unless the tag was long and of high certainty. The approach also derived more precise error rates on the analyzed combinatorial peptide library than those estimated using PeptideProphet and Percolator, showing its potential applicability for the detection of homologous peptides. Although the approach requires further computational developments for routine data analysis, it illustrates the value of peptide prior probabilities and presents a Bayesian approach for their incorporation into peptide detection.
- MeSH
- Algorithms MeSH
- Bayes Theorem MeSH
- Databases, Protein MeSH
- Peptide Library * MeSH
- Peptides * analysis MeSH
- Proteome analysis MeSH
- Proteomics MeSH
- Publication type
- Journal Article MeSH
We designed a combinatorial library of trifunctional scaffold-derived compounds, which were derivatized with 30 different in-house-made azides. The compounds were proposed to mimic insulin receptor (IR)-binding epitopes in the insulin molecule and bind to and activate this receptor. This work has enabled us to test our synthetic and biological methodology and to prove its robustness and reliability for the solid-phase synthesis and testing of combinatorial libraries of the trifunctional scaffold-derived compounds. Our effort resulted in the discovery of two compounds, which were able to weakly induce the autophosphorylation of IR and weakly bind to this receptor at a 0.1 mM concentration. Despite these modest biological results, which well document the well-known difficulty in modulating protein-protein interactions, this study represents a unique example of targeting the IR with a set of nonpeptide compounds that were specifically designed and synthesized for this purpose. We believe that this work can open new perspectives for the development of next-generation insulin mimetics based on the scaffold structure.
- MeSH
- Azides chemical synthesis chemistry MeSH
- Insulin analogs & derivatives chemistry metabolism MeSH
- Small Molecule Libraries chemical synthesis chemistry metabolism pharmacology MeSH
- Copper analysis MeSH
- Molecular Structure MeSH
- Receptor, Insulin chemistry metabolism MeSH
- Reproducibility of Results MeSH
- Combinatorial Chemistry Techniques * MeSH
- Solid-Phase Synthesis Techniques MeSH
- Protein Binding MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Publication type
- Journal Article MeSH
Described is a computer-assisted rational design of a DNA-bis-intercalator peptide library. The peptide library of 250 members was prepared and the most powerful binder identified. A value of the binding constant is almost two orders of magnitude higher than that of starting building block-9-aminoacridine. The binder affinity found toward calf thymus DNA is 30-fold of that of human prion peptide 106-126.
- MeSH
- Aminacrine chemistry MeSH
- Computer-Aided Design MeSH
- DNA-Binding Proteins chemistry metabolism MeSH
- DNA chemical synthesis chemistry metabolism MeSH
- Financing, Organized MeSH
- Fluorescent Dyes chemistry MeSH
- Gene Library MeSH
- Intercalating Agents chemistry MeSH
- Humans MeSH
- Models, Molecular MeSH
- Peptide Fragments metabolism MeSH
- Prions metabolism MeSH
- Cattle MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
- Combinatorial Chemistry Techniques MeSH
- Thymus Gland chemistry MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Cattle MeSH
- Animals MeSH
Galectin-3 (Gal-3), a member of the β-galactoside-binding lectin family, is a tumor biomarker and involved in tumor angiogenesis and metastasis. Gal-3 is therefore considered as a promising target for early cancer diagnosis and anticancer therapy. We here present the synthesis of a library of tailored multivalent neo-glycoproteins and evaluate their Gal-3 binding properties. By the combinatorial use of glycosyltransferases and chemo-enzymatic reactions, we first synthesized a set of N-acetyllactosamine (Galβ1,4GlcNAc; LacNAc type 2)-based oligosaccharides featuring five different terminating glycosylation epitopes, respectively. Neo-glycosylation of bovine serum albumin (BSA) was accomplished by dialkyl squarate coupling to lysine residues resulting in a library of defined multivalent neo-glycoproteins. Solid-phase binding assays with immobilized neo-glycoproteins revealed distinct affinity and specificity of the multivalent glycan epitopes for Gal-3 binding. In particular, neo-glycoproteins decorated with N',N″-diacetyllactosamine (GalNAcβ1,4GlcNAc; LacdiNAc) epitopes showed high selectivity and were demonstrated to capture Gal-3 from human serum with high affinity. Furthermore, neo-glycoproteins with terminal biotinylated LacNAc glycan motif could be utilized as Gal-3 detection agents in a sandwich enzyme-linked immunosorbent assay format. We conclude that, in contrast to antibody-based capture steps, the presented neo-glycoproteins are highly useful to detect functionally intact Gal-3 with high selectivity and avidity. We further gain novel insights into the binding affinity of Gal-3 using tailored multivalent neo-glycoproteins, which have the potential for an application in the context of cancer-related biomedical research.
- MeSH
- Amino Sugars chemical synthesis chemistry metabolism MeSH
- Galectin 3 antagonists & inhibitors metabolism MeSH
- Glycoproteins chemical synthesis chemistry metabolism pharmacology MeSH
- Glycosylation MeSH
- Humans MeSH
- Ligands MeSH
- Oligosaccharides chemical synthesis chemistry metabolism MeSH
- Serum Albumin, Bovine chemical synthesis chemistry metabolism pharmacology MeSH
- Cattle MeSH
- Combinatorial Chemistry Techniques MeSH
- Protein Binding MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Cattle MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
HotSpot Wizard 2.0 is a web server for automated identification of hot spots and design of smart libraries for engineering proteins' stability, catalytic activity, substrate specificity and enantioselectivity. The server integrates sequence, structural and evolutionary information obtained from 3 databases and 20 computational tools. Users are guided through the processes of selecting hot spots using four different protein engineering strategies and optimizing the resulting library's size by narrowing down a set of substitutions at individual randomized positions. The only required input is a query protein structure. The results of the calculations are mapped onto the protein's structure and visualized with a JSmol applet. HotSpot Wizard lists annotated residues suitable for mutagenesis and can automatically design appropriate codons for each implemented strategy. Overall, HotSpot Wizard provides comprehensive annotations of protein structures and assists protein engineers with the rational design of site-specific mutations and focused libraries. It is freely available at http://loschmidt.chemi.muni.cz/hotspotwizard.
- MeSH
- Automation MeSH
- Biocatalysis MeSH
- Databases, Protein MeSH
- Internet * MeSH
- Evolution, Molecular MeSH
- Models, Molecular MeSH
- Mutation * MeSH
- Mutagenesis, Site-Directed methods MeSH
- Peptide Library * MeSH
- Proteins chemistry genetics MeSH
- Software * MeSH
- Protein Stability MeSH
- Amino Acid Substitution MeSH
- Substrate Specificity MeSH
- Publication type
- Journal Article MeSH
Defining dynamic protein-protein interactions in the ubiquitin conjugation reaction is a challenging research area. Generating peptide aptamers that target components such as ubiquitin itself, E1, E2, or E3 could provide tools to dissect novel features of the enzymatic cascade. Next-generation deep sequencing platforms were used to identify peptide sequences isolated from phage-peptide libraries screened against Ubiquitin and its ortholog NEDD8. In over three rounds of selection under differing wash criteria, over 13,000 peptides were acquired targeting ubiquitin, while over 10,000 peptides were selected against NEDD8. The overlap in peptides against these two proteins was less than 5% suggesting a high degree in specificity of Ubiquitin or NEDD8 toward linear peptide motifs. Two of these ubiquitin-binding peptides were identified that inhibit both E3 ubiquitin ligases MDM2 and CHIP. NMR analysis highlighted distinct modes of binding of the two different peptide aptamers. These data highlight the utility of using next-generation sequencing of combinatorial phage-peptide libraries to isolate peptide aptamers toward a protein target that can be used as a chemical tool in a complex multi-enzyme reaction.
- Publication type
- Journal Article MeSH
The protein sequences found in nature represent a tiny fraction of the potential sequences that could be constructed from the 20-amino-acid alphabet. To help define the properties that shaped proteins to stand out from the space of possible alternatives, we conducted a systematic computational and experimental exploration of random (unevolved) sequences in comparison with biological proteins. In our study, combinations of secondary structure, disorder, and aggregation predictions are accompanied by experimental characterization of selected proteins. We found that the overall secondary structure and physicochemical properties of random and biological sequences are very similar. Moreover, random sequences can be well-tolerated by living cells. Contrary to early hypotheses about the toxicity of random and disordered proteins, we found that random sequences with high disorder have low aggregation propensity (unlike random sequences with high structural content) and were particularly well-tolerated. This direct structure content/aggregation propensity dependence differentiates random and biological proteins. Our study indicates that while random sequences can be both structured and disordered, the properties of the latter make them better suited as progenitors (in both in vivo and in vitro settings) for further evolution of complex, soluble, three-dimensional scaffolds that can perform specific biochemical tasks.
- MeSH
- Circular Dichroism MeSH
- Databases, Protein MeSH
- Datasets as Topic MeSH
- Models, Molecular * MeSH
- Nuclear Magnetic Resonance, Biomolecular MeSH
- Peptide Library * MeSH
- Protein Aggregates MeSH
- Recombinant Proteins chemistry isolation & purification toxicity MeSH
- Solubility MeSH
- Protein Folding MeSH
- Protein Structure, Secondary * MeSH
- Amino Acid Sequence MeSH
- Computational Biology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Single-chain antibodies (scFv) specific to Brachyspira hyodysenteriae were isolated from a phagemid library. Recombinant Bhlp 29.7 protein was used for scFv selection and individual clones were tested by ELISA and immunofluorescent test; four unique clones were isolated. One of selected clones was able to bind specifically B. hyodysenteriae in ELISA and immunofluorescence test. This is the first report of species-specific recombinant antibodies against B. hyodysenteriae.
- MeSH
- Antigens, Bacterial immunology MeSH
- Bacterial Proteins immunology MeSH
- Brachyspira hyodysenteriae immunology MeSH
- Species Specificity MeSH
- Enzyme-Linked Immunosorbent Assay MeSH
- Fluorescent Antibody Technique, Direct MeSH
- Gram-Negative Bacterial Infections diagnosis veterinary MeSH
- Lipoproteins immunology MeSH
- Antibodies, Monoclonal immunology MeSH
- Swine Diseases diagnosis MeSH
- Peptide Library MeSH
- Swine MeSH
- Antibodies, Bacterial immunology MeSH
- Recombinant Fusion Proteins immunology MeSH
- Antibody Specificity MeSH
- Immunoglobulin Variable Region immunology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
Recombinant ligands derived from small protein scaffolds show promise as robust research and diagnostic reagents and next generation protein therapeutics. Here, we derived high-affinity binders of human interferon gamma (hIFNγ) from the three helix bundle scaffold of the albumin-binding domain (ABD) of protein G from Streptococcus G148. Computational interaction energy mapping, solvent accessibility assessment, and in silico alanine scanning identified 11 residues from the albumin-binding surface of ABD as suitable for randomization. A corresponding combinatorial ABD scaffold library was synthesized and screened for hIFNγ binders using in vitro ribosome display selection, to yield recombinant ligands that exhibited K(d) values for hIFNγ from 0.2 to 10 nM. Molecular modeling, computational docking onto hIFNγ, and in vitro competition for hIFNγ binding revealed that four of the best ABD-derived ligands shared a common binding surface on hIFNγ, which differed from the site of human IFNγ receptor 1 binding. Thus, these hIFNγ ligands provide a proof of concept for design of novel recombinant binding proteins derived from the ABD scaffold.
- MeSH
- Bacterial Proteins chemistry genetics metabolism MeSH
- Gene Library MeSH
- Interferon-gamma metabolism MeSH
- Catalytic Domain MeSH
- Humans MeSH
- Models, Molecular MeSH
- Mutation MeSH
- Recombinant Proteins chemistry genetics metabolism MeSH
- Serum Albumin metabolism MeSH
- Streptococcus chemistry genetics metabolism MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Bacteriophage T4 is a virus with well-known genetics, structure, and biology. Such techniques as X-ray crystallography, cryo-EM, and three-dimensional (3D) image reconstruction allowed describing its structure very precisely. The genome of this bacteriophage was completely sequenced, which opens the way for the use of many molecular techniques, such as site-specific mutagenesis, which was widely applied, e.g., in investigating the functions of some essential T4 proteins. The phage-display method, which is commonly applied in bacteriophage modifications, was successfully used to display antigens (PorA protein, VP2 protein of vvIBDV, and antigens of anthrax and HIV) on T4's capsid platform. As first studies showed, the phage-display system as well as site-specific mutagenesis may also be used to modify interactions between phage particles and mammalian cells or to obtain phages infecting species other than the host bacteria. These may be used, among others, in the constantly developing bacteriophage therapy. All manipulations of this popular bacteriophage may enable the development of vaccine technology, phage therapy, and other branches of biological and medical science.
