MOTIVATION: Current techniques of protein engineering focus mostly on re-designing small targeted regions or defined structural scaffolds rather than constructing combinatorial libraries of versatile compositions and lengths. This is a missed opportunity because combinatorial libraries are emerging as a vital source of novel functional proteins and are of interest in diverse research areas. RESULTS: Here, we present a computational tool for Combinatorial Library Design (CoLiDe) offering precise control over protein sequence composition, length and diversity. The algorithm uses evolutionary approach to provide solutions to combinatorial libraries of degenerate DNA templates. We demonstrate its performance and precision using four different input alphabet distribution on different sequence lengths. In addition, a model design and experimental pipeline for protein library expression and purification is presented, providing a proof-of-concept that our protocol can be used to prepare purified protein library samples of up to 1011-1012 unique sequences. CoLiDe presents a composition-centric approach to protein design towards different functional phenomena. AVAILABILITYAND IMPLEMENTATION: CoLiDe is implemented in Python and freely available at https://github.com/voracva1/CoLiDe. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

• MeSH
• algoritmy * MeSH
• genová knihovna MeSH
• proteinové inženýrství MeSH
• proteiny * genetika   MeSH
• sekvence aminokyselin MeSH
• software MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny * MeSH

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.

• Klíčová slova
• Bayes’ theorem, Tandem mass spectrometry, combinatorial peptide library, peptide detection, prior probability,
• MeSH
• algoritmy MeSH
• Bayesova věta MeSH
• databáze proteinů MeSH
• peptidová knihovna * MeSH
• peptidy * analýza   MeSH
• proteom analýza   MeSH
• proteomika MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• peptidová knihovna * MeSH
• peptidy * MeSH
• proteom MeSH

The binding process of insulin to its transmembrane receptor entails a sophisticated interplay between two proteins, each possessing two binding sites. Given the difficulties associated with the use of insulin in the treatment of diabetes, despite its remarkable efficacy, there is interest in smaller and more stable compounds than the native hormone that would effectively activate the receptor. Our study adopts a strategy focused on synthesizing extensive combinatorial libraries of bipodal compounds consisting of two distinct peptides linked to a molecular scaffold. These constructs, evaluated in a resin bead-bound format, were designed to assess their binding to the insulin receptor. Despite notable nonspecific binding, our approach successfully generated and tested millions of compounds. Rigorous evaluations via flow cytometry and specific antibodies revealed peptide sequences with specific interactions at either receptor binding Site 1 or 2. Notably, these sequences bear similarity to peptides discovered through phage display by other researchers. This convergence of chemical and biological methods underscores nature's beauty, revealing general principles in peptide binding to the insulin receptor. Overall, our study deepens the understanding of molecular interactions in ligand binding to the insulin receptor, highlighting the challenges of targeting large proteins with small synthetic peptides.

• Klíčová slova
• insulin, library, peptide, receptor, scaffold,
• MeSH
• inzulin metabolismus   chemie   MeSH
• knihovny malých molekul chemie   farmakologie   chemická syntéza   MeSH
• lidé MeSH
• ligandy MeSH
• molekulární struktura MeSH
• peptidová knihovna MeSH
• peptidy chemie   metabolismus   chemická syntéza   MeSH
• receptor inzulinu * metabolismus   chemie   MeSH
• techniky kombinatorické chemie * MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• inzulin MeSH
• knihovny malých molekul MeSH
• ligandy MeSH
• peptidová knihovna MeSH
• peptidy MeSH
• receptor inzulinu * 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.

• Klíčová slova
• insulin mimetics, insulin receptor, library, protein-protein interactions, scaffold, trifunctional,
• MeSH
• azidy chemická syntéza   chemie   MeSH
• inzulin analogy a deriváty   chemie   metabolismus   MeSH
• knihovny malých molekul chemická syntéza   chemie   metabolismus   farmakologie   MeSH
• měď analýza   MeSH
• molekulární struktura MeSH
• receptor inzulinu chemie   metabolismus   MeSH
• reprodukovatelnost výsledků MeSH
• techniky kombinatorické chemie * MeSH
• techniky syntézy na pevné fázi MeSH
• vazba proteinů MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azidy MeSH
• inzulin MeSH
• knihovny malých molekul MeSH
• měď MeSH
• receptor inzulinu MeSH

A new method to find novel protein targets for ligands of interest is proposed. The principle of this approach is based on affinity chromatography and combinatorial chemistry. The proteins within a crude rat liver homogenate were allowed to interact with a combinatorial library of phosphinic pseudopeptides immobilized on affinity columns. Betaine: homocysteine S-methyltransferase (BHMT) was one of the proteins that was retained and subsequently eluted from these supports. The phosphinic pseudopeptides, which served as immobilized ligands for the isolation of rat BHMT, were then tested for their ability to inhibit human recombinant BHMT in solution. The most potent inhibitor also behaved as a selective ligand for the affinity purification of BHMT from a complex media. Further optimization uncovered Val-Phe-psi[PO(2-)-CH(2)]-Leu-His-NH(2) as a potent BHMT inhibitor that has an IC(50) of about 1 microM.

• MeSH
• betain-homocystein-S-methyltransferasa MeSH
• chromatografie afinitní * MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• enoyl-CoA-hydratasa antagonisté a inhibitory   MeSH
• glutathiontransferasa antagonisté a inhibitory   MeSH
• inhibitory enzymů chemická syntéza   farmakologie   MeSH
• játra metabolismus   MeSH
• krysa rodu Rattus MeSH
• kyseliny fosfinové chemická syntéza   MeSH
• lidé MeSH
• ligandy MeSH
• methyltransferasy antagonisté a inhibitory   MeSH
• molekulární sekvence - údaje MeSH
• peptidová knihovna MeSH
• peptidy chemie   izolace a purifikace   MeSH
• potkani Wistar MeSH
• rekombinantní proteiny chemická syntéza   farmakologie   MeSH
• sekvence aminokyselin MeSH
• techniky in vitro MeSH
• techniky kombinatorické chemie * MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• betain-homocystein-S-methyltransferasa MeSH
• BHMT protein, human MeSH Prohlížeč
• Bhmt protein, rat MeSH Prohlížeč
• enoyl-CoA-hydratasa MeSH
• glutathiontransferasa MeSH
• inhibitory enzymů MeSH
• kyseliny fosfinové MeSH
• ligandy MeSH
• methyltransferasy MeSH
• peptidová knihovna MeSH
• peptidy MeSH
• rekombinantní proteiny 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
• aminakrin chemie   MeSH
• design s pomocí počítače MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• DNA chemická syntéza   chemie   metabolismus   MeSH
• fluorescenční barviva chemie   MeSH
• genová knihovna * MeSH
• interkalátory chemie   MeSH
• lidé MeSH
• molekulární modely MeSH
• peptidové fragmenty metabolismus   MeSH
• priony metabolismus   MeSH
• skot MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• techniky kombinatorické chemie MeSH
• thymus chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• aminakrin MeSH
• DNA vazebné proteiny MeSH
• DNA MeSH
• fluorescenční barviva MeSH
• interkalátory MeSH
• peptidové fragmenty MeSH
• prion protein (106-126) MeSH Prohlížeč
• priony 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
• aminocukry chemická syntéza   chemie   metabolismus   MeSH
• galektin 3 antagonisté a inhibitory   metabolismus   MeSH
• glykoproteiny chemická syntéza   chemie   metabolismus   farmakologie   MeSH
• glykosylace MeSH
• lidé MeSH
• ligandy MeSH
• oligosacharidy chemická syntéza   chemie   metabolismus   MeSH
• sérový albumin hovězí chemická syntéza   chemie   metabolismus   farmakologie   MeSH
• skot MeSH
• techniky kombinatorické chemie MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminocukry MeSH
• galektin 3 MeSH
• glykoproteiny MeSH
• ligandy MeSH
• N-acetyllactosamine MeSH Prohlížeč
• oligosacharidy MeSH
• sérový albumin hovězí MeSH

The development of canine immunotolerant monoclonal antibodies can accelerate the invention of new medicines for both canine and human diseases. We develop a methodology to clone the naive, somatically mutated variable domain repertoire from canine B cell mRNA using 5'RACE PCR. A set of degenerate primers were then designed and used to clone variable domain genes into archival "holding" plasmid libraries. These archived variable domain genes were then combinatorially ligated to produce a scFv M13 phage library. Next-generation long-read and short-read DNA sequencing methodologies were developed to annotate features of the cloned library including CDR diversity and IGHV/IGKV/IGLV subfamily distribution. A synthetic immunoglobulin G was developed from this scFv library to the canine immune checkpoint receptor PD-1. This synthetic platform can be used to clone and annotate archived antibody variable domain genes for use in perpetuity in order to develop improved preclinical models for the treatment of complex human diseases.

• Klíčová slova
• CP: cancer biology, CP: immunology, canine physiology, next-generation sequencing, scfv phage libraries, translational research, veterinary medicine,
• MeSH
• jednořetězcové protilátky * genetika   imunologie   MeSH
• lidé MeSH
• monoklonální protilátky MeSH
• nádory * imunologie   MeSH
• peptidová knihovna * MeSH
• psi MeSH
• rekombinantní proteiny izolace a purifikace   imunologie   genetika   MeSH
• translační biomedicínský výzkum * metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• jednořetězcové protilátky * MeSH
• monoklonální protilátky MeSH
• peptidová knihovna * MeSH
• rekombinantní proteiny 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
• automatizace MeSH
• biokatalýza MeSH
• databáze proteinů MeSH
• internet * MeSH
• molekulární evoluce MeSH
• molekulární modely MeSH
• mutace * MeSH
• mutageneze cílená metody   MeSH
• peptidová knihovna * MeSH
• proteiny chemie   genetika   MeSH
• software * MeSH
• stabilita proteinů MeSH
• substituce aminokyselin MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• peptidová knihovna * MeSH
• proteiny 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.

• Klíčová slova
• aptamers, molecular dynamics, next-generation sequencing, phage-peptide, protein–peptide binding, ubiquitin,
• Publikační typ
• časopisecké články MeSH