Bioregulator from rat liver tissue

It has been shown that the membranotropic homeostatic tissue-specific bioregulator isolated from rat liver tissue contains a nanosized peptide-protein complex consisting of low-molecular peptides (1–6.5 kDa) and a protein from the serum albumin family. This bioregulator modulated the peptide biological activity and determined the tissue specificity.     

Antibodies against synthetic fragments of the prion protein for the diagnosis of bovine spongiform encephalopathy

Seven peptides matching fragments of the prion protein and containing from 17 to 31 amino acid residues were synthesized to obtain antibodies for diagnostics of bovine spongiform encephalopathy. Rabbits were immunized with either free peptides or peptide-protein conjugates to result in sera with a high level of antipeptide antibodies. Immunohistochemical assay revealed sera against four free peptides and a protein-peptide conjugate, which effectively bind to the pathogenic isoform of the prion protein in brain tissue preparations from cattle afflicted with bovine spongiform encephalopathy and do not interact with normal brain preparations. The resulting antipeptide sera can be used in developing a diagnostic kit for bovine spongiform encephalopathy.     

The Src SH2 domain interacts dynamically with the focal adhesion kinase binding site as demonstrated by paramagnetic NMR spectroscopy

The interaction between the tyrosine kinases Src and focal adhesion kinase (FAK) is a key step in signaling processes from focal adhesions. The phosphorylated tyrosine residue 397 in FAK is able to bind the Src SH2 domain. To establish the extent of the FAK binding motif, the binding affinity of the SH2 domain for phosphorylated and unphosphorylated FAK-derived peptides of increasing length was determined and compared with that of the internal Src SH2 binding site. It is shown that the FAK peptides have higher affinity than the internal binding site and that seven negative residues adjacent to the core SH2 binding motif increase the binding constant 30-fold. A rigid spin-label incorporated in the FAK peptides was used to establish on the basis of paramagnetic relaxation enhancement whether the peptide-protein complex is well defined. A large spread of the paramagnetic effects on the surface of the SH2 domain suggests that the peptide-protein complex exhibits dynamics, despite the high affinity of the peptide. The strong electrostatic interaction between the positive side of the SH2 domain and the negative peptide results in a high affinity but may also favor a dynamic interaction.     

EmPLiCS: an empirical approach for structure-based design of natural peptide drugs

The computer implementation of a peptide drug-design strategy has been developed. The system is named EmPLiCS (Empirical Peptide Ligand Construction System) according to the strategy of the system, which searches for peptide-ligand structures by referring to empirical rules that are derived from known protein 3D structures. The system was tested on several known peptide-protein complexes. The results demonstrated the ability of this system to detect key residues of peptides that are crucial for interaction with their specific proteins. The system also showed the ability to detect the main chain trace of these peptides. Some of the main chain atoms were detected even though the complete primary structures were not reproduced, suggesting that main chain structure is important in peptide-protein recognition. The results of the present study demonstrated that the empirical rules-based system can generate significant information for use in the design of natural peptide drugs.     

Microarrays of peptides elevated on the protein layer for efficient protein kinase assay

Peptide microarrays can be used for the high-throughput analysis of protein-peptide interactions. However, current peptide microarrays are rather costly to make and require cumbersome steps of introducing novel polymeric surfaces and/or chemical derivatization of peptides. Here, we report a novel method for manufacturing peptide microarrays by elevating the peptide on the layer of protein by a fusion protein approach. Using two protein kinases and their peptide substrates as examples, we show that elevating peptides on the layer of protein allows sensitive, specific, and efficient detection of peptide-protein interactions without the need for complicated chemical modification of solid supports and peptides. It was found that kinase activity could be detected with as low as 0.09 fmol of kemptide, which is about 1000-fold more sensitive than the 0.1 pmol obtained with other microarray systems. Furthermore, peptides can be produced as fusion proteins by fermentation of recombinant Escherichia coli and thus the expensive peptide synthesis process can be avoided. Therefore, this new strategy will not only be useful in high-throughput and cost-effective screening of kinase substrate peptides but also be generally applicable in studying various protein-peptide interactions.     

Improved two-dimensional reversed phase-reversed phase LC-MS/MS approach for identification of peptide-protein interactions

Quantitative mass spectrometry (MS) in combination with affinity purification approaches allows for an unbiased study of protein-protein and peptide-protein interactions. In shotgun approaches that are based on proteolytic digestion of complex protein mixtures followed by two-dimensional liquid-phase chromatography, the separation effort prior to MS analysis is focused on tryptic peptides. Here we developed an improved offline 2-D liquid chromatography-MS/MS approach for the identification and quantification of binding proteins utilizing reversed-phase capillary columns with acidic acetonitrile-containing eluents in both chromatographic dimensions. A specific fractionation scheme was applied in order to obtain samples with evenly distributed peptides and to fully utilize the separation space in the second dimension nanoLC-MS/MS. We report peptide-protein interaction studies to identify phosphorylation-dependent binding partners of the T cell adapter protein ADAP. The results of the SILAC-based pull-down experiments show this approach is well suited for distinguishing phosphorylation-specific interactions from unspecific binding events. The data provide further evidence that phosphorylated Tyr 595 of ADAP may serve as a direct binding site for the SH2 domains of the T cell proteins SLP76 and NCK. From a technical point of view we provide a detailed protocol for an offline 2-D RP-RP LC-MS/MS method that offers a robust and time-saving alternative for quantitative interactome analysis.     

Protein labeling and biotinylation of peptides during spot synthesis using biotin p-nitrophenyl ester (biotin-ONp)

Biotin-labeled peptides are used for numerous biochemical and microbiological applications. Due to the strong affinity of biotin to streptavidin, the detection of biotinylated molecules is very sensitive. A powerful technique for parallel synthesis and high-throughput screening of peptides is the spot synthesis. One example for the use of spot synthesis is the screening of biotinylated peptides synthesized on cellulose membranes, which is particularly favorable for the investigation of protease cleavage sites. Additionally, in combination with biotinylated protein samples, the spot technique can be used for investigations of peptide-protein and protein-protein interactions. Here, we present our results of the use biotin p-nitrophenyl ester (biotin-ONp) in spot synthesis and as a reagent for biotin-labeling of protein samples.     

Additive method for the prediction of protein-peptide binding affinity. Application to the MHC class I molecule HLA-A*0201

A method has been developed for prediction of binding affinities between proteins and peptides. We exemplify the method through its application to binding predictions of peptides with affinity to major histocompatibility complex class I molecule HLA-A*0201. The method is named "additive" because it is based on the assumption that the binding affinity of a peptide could be presented as a sum of the contributions of the amino acids at each position and the interactions between them. The amino acid contributions and the contributions of the interactions between adjacent side chains and every second side chain were derived using a partial least squares (PLS) statistical methodology using a training set of 420 experimental IC50 values. The predictive power of the method was assessed using rigorous cross-validation and using an independent test set of 89 peptides. The mean value of the residuals between the experimental and predicted pIC50 values was 0.508 for this test set. The additive method was implemented in a program for rapid T-cell epitope search. It is universal and can be applied to any peptide-protein interaction where binding data is known.     

Use of water-soluble 2-nitro-4-sulfophenyl ester of adipic acid for preparation of peptide protein conjugates]

Two synthetic peptides were conjugated with bovine serum albumin by means of 2-nitro-4-sulfophenyl ester of adipic acid. The amino acid analysis of the conjugates has shown that 14-15 molecules of the peptide are coupled per 1 molecule of the albumin during 10 min. The number of coupled molecules is the same when the reaction time increases to 24 hours. So, 2-nitro-4-sulfophenyl ester of adipic acid may be used parallel with the known bifunctional reagents to obtain peptide-protein conjugates.     

Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding.

We have solved the high-resolution X-ray structure of 14-3-3 bound to two different phosphoserine peptides, representing alternative substrate-binding motifs. These structures reveal an evolutionarily conserved network of peptide-protein interactions within all 14-3-3 isotypes, explain both binding motifs, and identify a novel intrachain phosphorylation-mediated loop structure in one of the peptides. A 14-3-3 mutation disrupting Raf signaling alters the ligand-binding cleft, selecting a different phosphopeptide-binding motif and different substrates than the wild-type protein. Many 14-3-3: peptide contacts involve a C-terminal amphipathic alpha helix containing a putative nuclear export signal, implicating this segment in both ligand and Crm1 binding. Structural homology between the 14-3-3 NES structure and those within I kappa B alpha and p53 reveals a conserved topology recognized by the Crm1 nuclear export machinery.     

Electron transfer reactions of metalloproteins at peptide-modified gold electrodes

The electron transfer reactions of four small redox proteins, cytochrome c. ferredoxin, plastocyanin and azurin, have been investigated at novel peptide-modified gold electrodes. These proved to be effective and selective in facilitating electron transfer. Good, quasi-reversible electron transfer was achieved selectively at different peptide-protein configurations by changing the pH or the ionic strength of the solution. The use of peptides as promoters for protein electrochemistry opens up the possibility of designing very specific electrode surfaces for larger molecules like enzymes.     

Enhanced translocation of amphiphilic peptides across membranes by transmembrane proteins

Cell membranes are phospholipid bilayers with a large number of embedded transmembrane proteins. Some of these proteins, such as scramblases, have properties that facilitate lipid flip-flop from one membrane leaflet to another. Scramblases and similar transmembrane proteins could also affect the translocation of other amphiphilic molecules, including cell-penetrating or antimicrobial peptides. We studied the effect of transmembrane proteins on the translocation of amphiphilic peptides through the membrane. Using two very different models, we consistently demonstrate that transmembrane proteins with a hydrophilic patch enhance the translocation of amphiphilic peptides by stabilizing the peptide in the membrane. Moreover, there is an optimum amphiphilicity because the peptide could become overstabilized in the transmembrane state, in which the peptide-protein dissociation is hampered, limiting the peptide translocation. The presence of scramblases and other proteins with similar properties could be exploited for more efficient transport into cells. The described principles could also be utilized in the design of a drug-delivery system by the addition of a translocation-enhancing peptide that would integrate into the membrane.     

Peptide-protein complex from cattle sclera: Structural aspects and chaperone activity

The influence of temperature and chaotropic agents on the spatial organization of the peptide-protein complex isolated from cattle sclera at the level of secondary structure was studied by UV, CD spectroscopy, and dynamic light scattering. It is shown that this complex has high conformational thermostability. The point of conformational thermal transition (65 °C) was determined, after which the peptide-protein complex passes into a denatured stable state. It was found that the peptide-protein complex in aqueous solutions forms thermostable nanosized particles. It was shown that the peptide-protein complex isolated from cattle sclera shows the properties of chaperone, an inhibitor of model protein aggregation induced by dithiothreitol.     

The analysis of peptide affinity and its binding kinetics to DR1DW1 major histocompatibility complex protein.

The connection between experimentally measured values of ED50 (concentration of added peptide required to bind half of the protein), which characterize peptide-protein binding and the equilibrium dissociation constant of peptide-protein complex Kd (affinity) is considered. It is shown and confirmed by experimental studies that in certain cases, as a result of the absence of equilibrium in the system, the value of Kd could be much less than the experimental value of ED50, but not equal to that as commonly assumed. This is especially applicable to the formation of peptide-MHC complexes with low dissociation rates (strong binding), which may require longer time-intervals to reach equilibrium. Thus the search of the good binding peptides based on finding ones with the smallest measured values' of ED50 may result in missing the best binders with the lowest values of dissociation constant (highest affinity). To analyze the problem we considered the formal chemical kinetics of peptide-protein binding. Experimental studies of peptide binding was performed to obtain the parameters of the kinetic model. According to the predictions of the model, it was confirmed that peptide binding occurs through the preceding step, which is either a release of an endogenous peptide or some conformational change of the molecule. The half decay time for this process was determined to be approximately 3 h. Based on the model developed, a new effective method for determination of the dissociation rates of peptide-MHC complexes and the equilibrium dissociation constants Kd was proposed, which implies the comparison of binding levels (ED50) at different instants of time. This method works especially well for the peptide-MHC complexes with relatively slow dissociation rates (stable complexes), for which the direct off-rate measurements as well as obtaining equilibrium binding data to determine Kd are highly time consuming and not very reliable.     

Functional analysis of expressed peptides that bind yeast STE proteins

Peptides are potentially useful for target validation and other reverse genetic applications. For instance, if a specific protein is susceptible to peptide inhibition, it may have a higher probability of being vulnerable to small molecules. We used the yeast two-hybrid technique to identify and study peptide binders for three yeast proteins involved in pheromone response: Ste11p, Ste18p, and Ste50p. A subset of peptide binders was shown to inhibit pheromone response in cells using two different functional assays. In addition, we utilized a variant of the yeast two-hybrid method to examine relative binding affinities based on competitive interactions in yeast. Our results suggest that binding affinity and inhibitory potency of peptides do not correlate perfectly and that peptide-protein interactions can be complex and unpredictable. Taken together these results suggest that while peptides are useful as in vivo inhibitors of protein function, caution must be exercised when choosing peptides for further studies and when inferring affinities from expression phenotypes.     

A workflow for peptide-based proteomics in a poorly sequenced plant: a case study on the plasma membrane proteome of banana

Membrane proteins are an interesting class of proteins because of their functional importance. Unfortunately their analysis is hampered by low abundance and poor solubility in aqueous media. Since shotgun methods are high-throughput and partly overcome these problems, they are preferred for membrane proteomics. However, their application in non-model plants demands special precautions to prevent false positive identification of proteins. In the current paper, a workflow for membrane proteomics in banana, a poorly sequenced plant, is proposed. The main steps of this workflow are (i) optimization of the peptide separation, (ii) performing de novo sequencing to allow a sequence homology search and (iii) visualization of identified peptide-protein associations using Cytoscape to remove redundancy and wrongly assigned peptides, based on species-specific information. By applying this workflow, integral plasma membrane proteins from banana leaves were successfully identified.     

An Integrated Peptide-Antigen Microarray on Plasmonic Gold Films for Sensitive Human Antibody Profiling

High-throughput screening for interactions of peptides with a variety of antibody targets could greatly facilitate proteomic analysis for epitope mapping, enzyme profiling, drug discovery and biomarker identification. Peptide microarrays are suited for such undertaking because of their high-throughput capability. However, existing peptide microarrays lack the sensitivity needed for detecting low abundance proteins or low affinity peptide-protein interactions. This work presents a new peptide microarray platform constructed on nanostructured plasmonic gold substrates capable of metal enhanced NIR fluorescence enhancement (NIR-FE) by hundreds of folds for screening peptide-antibody interactions with ultrahigh sensitivity. Further, an integrated histone peptide and whole antigen array is developed on the same plasmonic gold chip for profiling human antibodies in the sera of systemic lupus erythematosus (SLE) patients, revealing that collectively a panel of biomarkers against unmodified and post-translationally modified histone peptides and several whole antigens allow more accurate differentiation of SLE patients from healthy individuals than profiling biomarkers against peptides or whole antigens alone.     

Modeling beta-sheet peptide-protein interactions: Rosetta FlexPepDock in CAPRI rounds 38-45

Peptide-protein docking is challenging due to the considerable conformational freedom of the peptide. CAPRI rounds 38-45 included two peptide-protein interactions, both characterized by a peptide forming an additional beta strand of a beta sheet in the receptor. Using the Rosetta FlexPepDock peptide docking protocol we generated top-performing, high-accuracy models for targets 134 and 135, involving an interaction between a peptide derived from L-MAG with DLC8. In addition, we were able to generate the only medium-accuracy models for a particularly challenging target, T121. In contrast to the classical peptide-mediated interaction, in which receptor side chains contact both peptide backbone and side chains, beta-sheet complementation involves a major contribution to binding by hydrogen bonds between main chain atoms. To establish how binding affinity and specificity are established in this special class of peptide-protein interactions, we extracted PeptiDBeta, a benchmark of solved structures of different protein domains that are bound by peptides via beta-sheet complementation, and tested our protocol for global peptide-docking PIPER-FlexPepDock on this dataset. We find that the beta-strand part of the peptide is sufficient to generate approximate and even high resolution models of many interactions, but inclusion of adjacent motif residues often provides additional information necessary to achieve high resolution model quality.     

Acceleration of protein aggregation by amphiphilic peptides: transformation of supramolecular structure of the aggregates

Protein self-assembly and aggregation represent a special tool in biomedicine and biotechnology to produce biological materials for a wide range of applications. The protein aggregates are very different morphologically, varying from soluble amorphous aggregates to highly ordered amyloid-like fibrils, the latter being associated with molecular structures able to perform specific functions in living systems. Fabrication of novel biomaterials resembling natural protein assemblies has awakened interest in identification of low-molecular-weight biogenic agents as regulators of transformation of aggregation-prone proteins into fibrillar structures. Short amphiphilic peptides can be considered for this role. Using dynamic light scattering, turbidimetry, fluorescence spectroscopy, and transmission electron microscopy (TEM), we have demonstrated that the Arg-Phe dipeptide dramatically accelerates the aggregation of a model protein, α-lactalbumin, to generate morphologically different structures. TEM revealed transformation of spherical particles observed in the control samples into branched chains of fibril-like nanostructures in the presence of the peptide, suggesting that amphiphilic peptides can induce changes in the physicochemical properties of a protein substrate (net charge, hydrophobicity, and tendency to β-structure formation) resulting in accumulation of peptide-protein complexes competent to self-assembly into supramolecular structures. A number of other short amphiphilic peptides have also been shown to accelerate the aggregation process, using alternative complementary protein substrates for identification of molecular recognition modules. Peptide-protein assemblies are suggested to play the role of building blocks for formation of supramolecular structures profoundly differing from those of the individual protein substrate in type, size, and shape.