Comparison of mRNA-display-based selections using synthetic peptide and natural protein libraries

mRNA display is a genotype-phenotype conjugation method that allows the amplification-based, iterative rounds of in vitro selection to be applied to peptides and proteins. Compared to prior protein selection techniques, mRNA display can be used to select functional sequences from both long natural protein and short combinatorial peptide libraries with much higher complexities. To investigate the basic features and problems of using mRNA display in studying conditional protein-protein interactions, we compared the target-binding selections against calmodulin (CaM) using both a natural protein library and a combinatorial peptide library. The selections were efficient in both cases and required only two rounds to isolate numerous Ca2+/CaM-binding natural proteins and synthetic peptides with a wide range of affinities. Many known and novel CaM-binding proteins were identified from the natural human protein library. More than 2000 CaM-binding peptides were selected from the combinatorial peptide library. Unlike sequences from prior CaM-binding selections that correlated poorly with naturally occurring proteins, synthetic peptides homologous to the Ca2+/CaM-binding motifs in natural proteins were isolated. Interestingly, a large number of synthetic peptides that lack the conventional CaM-binding secondary structures bound to CaM tightly and specifically, suggesting the presence of other interaction modes between CaM and its downstream binding targets. Our results indicate that mRNA display is an ideal approach to the identification of Ca2+-dependent protein-protein interactions, which are important in the regulation of numerous signaling pathways.     

Convergent evolution with combinatorial peptides

Once the sequence of a genome is in hand, understanding the function of its encoded proteins becomes a task of paramount importance. Much like the biochemists who first outlined different biochemical pathways, many genomic scientists are engaged in determining which proteins interact with which proteins, thereby establishing a protein interaction network. While these interactions have evolved in regard to their specificity, affinity and cellular function over billions of years, it is possible in the laboratory to isolate peptides from combinatorial libraries that bind to the same proteins with similar specificity, affinity and primary structures, which resemble those of the natural interacting proteins. We have termed this phenomenon 'convergent evolution'. In this review, we highlight various examples of convergent evolution that have been uncovered in experiments dissecting protein-protein interactions with combinatorial peptides. Thus, a fruitful approach for mapping protein-protein interactions is to isolate peptide ligands to a target protein and identify candidate interacting proteins in a sequenced genome by computer analysis.     

Peptide arrays with designed secondary structures for protein characterization using fluorescent fingerprint patterns

To realize a practical high-throughput protein-detection system, novel peptide arrays have been constructed using designed peptide libraries with loop, alpha-helix, or beta-strand structures. Here, we describe the overview of the reported designed peptide arrays with loop and alpha-helix structures and the new results of those with beta-strand structures. Initially, several model peptides known to interact with model structured proteins were selected to establish the present strategy for high-throughput detection of proteins. The fluorescent probes and suitable scaffolds of peptides were examined for the effective detection of proteins. The detection methods were established in solution and in an immobilized manner using the model systems. In the case of alpha-helix peptide, the response of a peptide with fluorescent resonance energy transfer between two probes at both termini was several times higher than that of a peptide with a single probe. In the cases of peptides with other structures, however, proteins were effectively detectable even by the fluorescent change of one probe. Furthermore, structurally focused libraries consisting of a total of ca. 250 different peptides based on the model peptides with secondary and/or tertiary structures were constructed with systematic replacement of residues. Using these libraries, various proteins were characterized effectively to give their own fluorescent "protein fingerprint" patterns. The resulting protein fingerprints correlated with the recognition properties of the proteins. These studies demonstrate that arrays with peptide libraries based on designed structures can be promising tools for detecting the target proteins. Designed synthetic peptides play roles as the capturing agents to be developed for practical protein chips.     

Mapping protein-protein interactions with combinatorial biology methods

Extremely diverse, DNA-encoded libraries of peptides and proteins have been constructed that include a linkage between each polypeptide and the encoding DNA. Library members can be selected by virtue of a particular binding specificity, and their protein sequence can be deduced from the sequence of the cognate DNA. Such combinatorial biology methods have proven invaluable in both identifying natural protein-protein interactions and also in mapping the specificities and energetics of these interactions in fine detail.     

Peptide signals encode protein localization

Many bacterial proteins are localized to precise intracellular locations, but in most cases the mechanism for encoding localization information is not known. Screening libraries of peptides fused to green fluorescent protein identified sequences that directed the protein to helical structures or to midcell. These peptides indicate that protein localization can be encoded in 20-amino-acid peptides instead of complex protein-protein interactions and raise the possibility that the location of a protein within the cell could be predicted from bioinformatic data.     

Expression,purification, and characterization of proteins from high-quality combinatorial libraries of the mammalian calmodulin central linker

Combinatorial libraries offer an attractive approach towards exploring protein sequence, structure and function. Although several strategies introduce sequence diversity, the likelihood of identifying proteins with novel functions is increased when the library of genes encodes for folded and soluble structures. Here we present the first application of the binary patterning approach of combinatorial protein library design to the unique central linker region of the highly-conserved protein, calmodulin (CaM). We show that this high-quality approach translates very well to the CaM protein scaffold: All library members over-express and are functionally diverse, having a range of conformations in the presence and absence of calcium as determined by circular dichroism spectroscopy. Collectively, these data support that the binary patterning approach, when applied to the highly-conserved protein fold, can yield large collections of folded, soluble and highly-expressible proteins.     

Natural product-like libraries based on non-aromatic, polycyclic motifs

Diversity-oriented synthesis is an intriguing approach for creating structurally diverse compounds that cover the pharmaceutically relevant chemical space in an optimal way. On the other hand, an over-proportionally large number of drugs or lead structures originate from compounds isolated from natural sources. Thus, not surprisingly, an increasing number of combinatorial libraries are based on motifs resembling natural products. A particular aspect of many natural products is the presence of non-aromatic, polycyclic core structures. The fusion of several rings leads to geometrically well-defined structures and, thus, holds the promise of a high functional specialisation. In this review we present several actual examples of natural product-like libraries with non-aromatic polycyclic motifs based on naturally occurring compounds.     

All D-amino acid hexapeptide inhibitors of melittin's cytolytic activity derived from synthetic combinatorial libraries

The identification of peptides that inhibit the biological functions of proteins was used as a means to explore protein/ligand interactions involved in molecular recognition processes. This approach is based on the use of synthetic combinatorial libraries (SCLs) for the rapid identification of individual peptides that block the interaction of proteins with their biological targets. Thus, each peptide mixture of an all-D -amino acid hexapeptide SCL in a positional scanning format was screened for its ability to inhibit the hemolytic activity of melittin, a model self-assembling protein. The potent inhibitory activity of the identified individual peptides suggests that protein-like complexes are able to specifically bind to peptides having an all-D configuration. These results also show that SCLs are useful for the identification of short, non-hydrolysable sequences having potential intracellular inhibitory activities.     

Mapping signal transduction pathways by phage display

Rapid identification of proteins that interact with a novel gene product is an important element of functional genomics. Here we describe a phage display-based technique for interaction screening of complex cDNA libraries using proteins or synthetic peptides as baits. Starting with the epidermal growth factor receptor (EGFR) cytoplasmic tail, we identified known protein interactions that link EGFR to the Ras/MAP kinase signal transduction cascade and several novel interactions. This approach can be used as a rapid and efficient tool for elucidating protein networks and mapping intracellular signal transduction pathways.     

Construction and application of a yeast surface-displayed human cDNA library to identify post-translational modification-dependent protein-protein interactions

Although post-translational modifications such as phosphorylation mediate fundamental biological processes within the cell, relatively few methods exist that allow proteome-wide identification of proteins that interact with these modifications. We constructed a yeast surface-displayed human cDNA library and utilized it to identify protein fragments with affinity for phosphorylated peptides derived from the major tyrosine autophosphorylation sites of the epidermal growth factor receptor or focal adhesion kinase. We identified cDNAs encoding the Src homology 2 domains from adapter protein APS, phosphoinositide 3-kinase regulatory subunit 3, SH2B, and tensin, demonstrating the effectiveness of this approach. Our results suggest that large libraries of functional human protein fragments can be efficiently displayed on the yeast surface. In addition to the analysis of post-translational modifications, yeast surface-displayed human cDNA libraries have many potential applications, including identifying targets and defining potential cross-reactive proteins for small molecules or drugs.     

mRNA display: ligand discovery,interaction analysis and beyond

In vitro peptide and protein selection using mRNA display enables the discovery and directed evolution of new molecules from combinatorial libraries. These selected molecules can serve as tools to control and understand biological processes, enhance our understanding of molecular interactions and potentially treat disease in therapeutic applications. In mRNA display, mRNA molecules are covalently attached to the peptide or protein they encode. These mRNA-protein fusions enable in vitro selection of peptide and protein libraries of >10(13) different sequences. mRNA display has been used to discover novel peptide and protein ligands for RNA, small molecules and proteins, as well as to define cellular interaction partners of proteins and drugs. In addition, several unique applications are possible with mRNA display, including self-assembling protein chips and library construction with unnatural amino acids and chemically modified peptides.     

In vivo application of photocleavable protein interaction reporter technology

In vivo protein structures and protein-protein interactions are critical to the function of proteins in biological systems. As a complementary approach to traditional protein interaction identification methods, cross-linking strategies are beginning to provide additional data on protein and protein complex topological features. Previously, photocleavable protein interaction reporter (pcPIR) technology was demonstrated by cross-linking pure proteins and protein complexes and the use of ultraviolet light to cleave or release cross-linked peptides to enable identification. In the present report, the pcPIR strategy is applied to Escherichia coli cells, and in vivo protein interactions and topologies are measured. More than 1600 labeled peptides from E. coli were identified, indicating that many protein sites react with pcPIR in vivo. From those labeled sites, 53 in vivo intercross-linked peptide pairs were identified and manually validated. Approximately half of the interactions have been reported using other techniques, although detailed structures exist for very few. Three proteins or protein complexes with detailed crystallography structures are compared to the cross-linking results obtained from in vivo application of pcPIR technology.     

Conformational analysis of invariant peptide sequences in bacterial genomes

The functional significance of evolutionarily conserved motifs/patterns of short regions in proteins is well documented. Although a large number of sequences are conserved, only a small fraction of these are invariant across several organisms. Here, we have examined the structural features of the functionally important peptide sequences, which have been found invariant across diverse bacterial genera. Ramachandran angles (phi,psi) have been used to analyze the conformation, folding patterns and geometrical location (buried/exposed) of these invariant peptides in different crystal structures harboring these sequences. The analysis indicates that the peptides preferred a single conformation in different protein structures, with the exception of only a few longer peptides that exhibited some conformational variability. In addition, it is noticed that the variability of conformation occurs mainly due to flipping of peptide units about the virtual C(alpha)...C(alpha) bond. However, for a given invariant peptide, the folding patterns are found to be similar in almost all the cases. Over and above, such peptides are found to be buried in the protein core. Thus, we can safely conclude that these invariant peptides are structurally important for the proteins, since they acquire unique structures across different proteins and can act as structural determinants (SD) of the proteins. The location of these SD peptides on the protein chain indicated that most of them are clustered towards the N-terminal and middle region of the protein with the C-terminal region exhibiting low preference. Another feature that emerges out of this study is that some of these SD peptides can also play the roles of "fold boundaries" or "hinge nucleus" in the protein structure. The study indicates that these SD peptides may act as chain-reversal signatures, guiding the proteins to adopt appropriate folds. In some cases the invariant signature peptides may also act as folding nuclei (FN) of the proteins.     

Advantages of mRNA display selections over other selection techniques for investigation of protein–protein interactions

mRNA display is a genotype–phenotype conjugation method that allows for amplification-based, iterative rounds of in vitro selection to be applied to peptides and proteins. mRNA display can be used to display both long natural protein and short synthetic peptide libraries with unusually high diversities for the investigation of protein–protein interactions. Here, we summarize the advantages of mRNA display by comparing it with other widely used peptide or protein-selection techniques, and discuss various applications of this technique in studying protein–protein interactions.     

Biotin-tagged cDNA expression libraries displayed on lambda phage: a new tool for the selection of natural protein ligands

cDNA expression libraries displayed on lambda phage have been successfully employed to identify partners involved in antibody–antigen, protein– protein and DNA–protein interactions and represent a novel approach to functional genomics. However, as in all other cDNA expression libraries based on fusion to a carrier polypeptide, a major issue of this system is the absence of control over the translation frame of the cDNA. As a consequence, a large number of clones will contain lambda D/cDNA fusions, resulting in the foreign sequence being translated on alternative reading frames. Thus, many phage will not display natural proteins, but could be selected, as they mimic the binding properties of the real ligand, and will hence interfere with the selection outcome. Here we describe a novel lambda vector for display of exogenous peptides at the C-terminus of the capsid D protein. In this vector, translation of fusion peptides in the correct reading frame allows efficient in vivo biotinylation of the chimeric phage during amplification. Using this vector system we constructed three libraries from human hepatoma cells, mouse hepatocytic MMH cells and from human brain. Clones containing open reading frames (ORFs) were rapidly selected by streptavidin affinity chromatography, leading to biological repertoires highly enriched in natural polypeptides. We compared the selection outcome of two independent experiments performed using an anti-GAP-43 monoclonal antibody on the human brain cDNA library before and after ORF enrichment. A significant increase in the efficiency of identification of natural target peptides with very little background of false-positive clones was observed in the latter case.     

Mass spectrometry-based shotgun glycomics for discovery of natural ligands of glycan-binding proteins

The non-covalent associations of complex carbohydrates (glycans) with glycan-binding proteins mediate many important physiological and pathophysiological processes. Identifying these interactions is essential to understanding their diverse biological functions and enables the development of new disease treatments and diagnostics. Knowledge of the repertoire of glycans recognized by most glycan-binding proteins and their affinities is incomplete. Mass spectrometry-based screening of natural glycan libraries has emerged as a promising approach to defining the glycan interactome of glycan-binding proteins. Here, we review recent advances in mass spectrometry-based natural library screening that have led to the discovery of glycan ligands of endogenous and exogenous proteins and illuminated their binding specificities.     

Interaction between Intrinsically Disordered Proteins Frequently Occurs in a Human Protein-Protein Interaction Network

Intrinsic protein disorder is a widespread phenomenon characterised by a lack of stable three-dimensional structures and is considered to play an important role in protein-protein interactions (PPIs). This study examined the genome-wide preference of disorder in PPIs by using exhaustive disorder prediction in human PPIs. We categorised the PPIs into three types (interaction between disordered proteins, interaction between structured proteins, and interaction between a disordered protein and a structured protein) with regard to the flexibility of molecular recognition and compared these three interaction types in an existing human PPI network with those in a randomised network. Although the structured regions were expected to become the identifiers for binding recognition, this comparative analysis revealed unexpected results. The occurrence of interactions between disordered proteins was significantly frequent, and that between a disordered protein and a structured protein was significantly infrequent. We found that this propensity was much stronger in interactions between nonhub proteins. We also analysed the interaction types from a functional standpoint by using GO, which revealed that the interaction between disordered proteins frequently occurred in cellular processes, regulation, and metabolic processes. The number of interactions, especially in metabolic processes between disordered proteins, was 1.8 times as large as that in the randomised network. Another analysis conducted by using KEGG pathways provided results where several signaling pathways and disease-related pathways included many interactions between disordered proteins. All of these analyses suggest that human PPIs preferably occur between disordered proteins and that the flexibility of the interacting protein pairs may play an important role in human PPI networks.     

Designed armadillo repeat proteins as general peptide-binding scaffolds: consensus design and computational optimization of the hydrophobic core

Armadillo repeat proteins are abundant eukaryotic proteins involved in several cellular processes, including signaling, transport, and cytoskeletal regulation. They are characterized by an armadillo domain, composed of tandem armadillo repeats of approximately 42 amino acids, which mediates interactions with peptides or parts of proteins in extended conformation. The conserved binding mode of the peptide in extended form, observed for different targets, makes armadillo repeat proteins attractive candidates for the generation of modular peptide-binding scaffolds. Taking advantage of the large number of repeat sequences available, a consensus-based approach combined with a force field-based optimization of the hydrophobic core was used to derive soluble, highly expressed, stable, monomeric designed proteins with improved characteristics compared to natural armadillo proteins. These sequences constitute the starting point for the generation of designed armadillo repeat protein libraries for the selection of peptide binders, exploiting their modular structure and their conserved binding mode.     

Defense peptides of plant immune system

Antimicrobial peptides (AMPs) are natural antibiotics produced by all living organisms to combat pathogens. They are important effector molecules of the immune system both in animals and plants. AMPs are diverse in structure and mode of action. Based on homology of amino acid sequences and 3D structures several AMP families have been distinguished. They are defensins, thionins, lipid transfer proteins, hevein- and knottin-like peptides, and cyclotides. AMPs display broad-spectrum antimicrobial activity and thus show promise for the development of disease- resistant crops by genetic engineering and for the production of new-generation drugs. In this paper, the properties of the main AMP families (defensins and hevein-like peptides) and of a new 4-Cys plant AMP family are reviewed.     

Mapping discontinuous protein‐binding sites via structure‐based peptide libraries: combining in silico and in vitro approaches

To perform their various functions, protein surfaces often have to interact with each other in a specific way. Usually, only parts of a protein are accessible and can act as binding sites. Because proteins consist of polypeptide chains that fold into complex three‐dimensional shapes, binding sites can be divided into two different types: linear sites that follow the primary amino acid sequence and discontinuous binding sites, which are made up of short peptide fragments that are adjacent in spatial proximity. Such discontinuous binding sites dominate protein–protein interactions, but are difficult to identify. To meet this challenge, we combined a computational, structure‐based approach and an experimental, high‐throughput method. SUPERFICIAL is a program that uses protein structures as input and generates peptide libraries to represent the protein's surface. A large number of the predicted peptides can be simultaneously synthesised applying the SPOT technology. The results of a binding assay subsequently help to elucidate protein–protein interactions; the approach is applicable to any kind of protein. The crystal structure of the complex of hen egg lysozyme with the well‐characterised murine IgG1 antibody HyHEL‐5 is available, and the complex is known to have a discontinuous binding site. Using SUPERFICIAL, the entire surface of lysozyme was translated into a peptide library that was synthesised on a cellulose membrane using the SPOT technology and tested against the HyHEL‐5 antibody. In this way, it was possible to identify two peptides (longest common sequence and peptide 19) that represented the discontinuous epitope of lysozyme. Copyright © 2012 John Wiley & Sons, Ltd.