Streptococcus mutans, a common oral pathogen and the causative agent of dental caries, has persisted and even thrived on the tooth surface despite constant removal and eradication efforts. In this study, we generated a number of synthetic antimicrobial peptides against this bacterium via construction and screening of several structurally diverse peptide libraries where the hydrophobicity and charge within each library was varied incrementally in order to generate a collection of peptides with different biochemical characteristics. From these libraries, we identified multiple peptides with robust killing activity against S. mutans. To further improve their effectiveness, the most bactericidal peptides from each library were synthesized together as one molecule, in various combinations, with and without a flexible peptide linker between each antimicrobial region. Many of these “fusion” peptides had enhanced killing activities in comparison with those of the original nonconjoined molecules. The results presented here illustrate that small libraries of biochemically constrained peptides can be used to generate antimicrobial peptides against S. mutans, several of which may be likely candidates for the development of anticaries agents.
Prolactin receptor is involved in normal lactation and reproduction; however, excessive prolactin levels can cause various reproductive disorders such as prolactinomas. Small-molecule antagonists against the human prolactin receptor (hPRLr) thus have potential clinical applications and may serve as useful molecular probes in biomedical research. In this work, we synthesized a large, support-bound cyclic peptide library (theoretical diversity of 1.2 x 107) on 90-μm TentaGel beads and screened it against the extracellular domain of hPRLr. To facilitate hit identification, each TentaGel bead was spatially segregated into outer and inner layers, with a cyclic peptide displayed on the bead surface while the bead interior contained the corresponding linear peptide. The identity of a positive bead was revealed by sequencing the linear encoding peptide within the bead by partial Edman degradation/mass spectrometry. Screening of the library resulted in 20 hits, two of which were selected for further analysis and shown to bind to hPRLr with dissociation constants of 2–3 μM.
Cyclic peptides; combinatorial library; partial Edman degradation; prolactin; prolactin receptor
Through glycosphingolipid biochemical research, we developed two types of transcription technologies. One is a biochemical transfer of glycosphingolipids to peptides. The other is a physicochemical transfer of glycosphingolipids in silica gel to the surface of a plastic membrane. Using the first technology, we could prepare peptides which mimic the shapes of glycosphingolipid molecules by biopanning with a phage-displayed peptide library and anti-glycosphingolipid antibodies as templates. The peptides thus obtained showed biological properties and functions similar to those of the original glycosphingolipids, such as lectin binding, glycosidase modulation, inhibition of tumor metastasis and immune response against the original antigen glycosphingolipid, and we named them glyco-replica peptides. The results showed that the newly prepared peptides could be used effectively as a bio-recognition system and suggest that the glyco-replica peptides can be widely applied to therapeutic fields. Using the second technology, we could establish a functional lipidomics with a thin-layer chromatography-blot/matrix-assisted laser desorption ionization-time of flight mass spectrometry (TLC-Blot/MALDI-TOF MS) system. By transferring glycosphingolipids on a plastic membrane surface from a TLC plate, innovative biochemical approaches such as simple purification of individual glycosphingolipids, binding studies, and enzyme reactions could be developed. The combinations of these biochemical approaches and MALDI-TOF MS on the plastic membrane could provide new strategies for glycosphingolipid science and the field of lipidomics. In this review, typical applications of these two transfer technologies are introduced.
glycosphingolipid; gangliosides; TLC-Blot/MALDI-TOF MS; phage-displayed peptide library; glycolipidomics; molecular mimicry
Tumor vasculature expresses a distinct set of molecule signatures on the endothelial cell surface different from the resting blood vessels of other organs and tissues in the body. This makes them an attractive target for cancer therapy and molecular imaging. The current technology using the in vivo phage display biopanning allows us to quickly isolate and identify peptides potentially homing to various tumor blood vessels. Tumor-homing peptides in conjugation with chemotherapeutic drugs or imaging contrast have been extensively tested in various preclinical and clinical studies. These tumor-homing peptides have valuable potential as targeting probes for tumor molecular imaging and drug delivery. In this review, we summarize the recent advances about the applications of tumor-homing peptides selected by in vivo phage display library screening against tumor vasculature. We also introduce the characteristics of the latest discovered tumor-penetrating peptides in their potential clinical applications.
Systematic identification of binding partners for modular domains such as Src homology 2 (SH2) is important for understanding the biological function of the corresponding SH2 proteins. We have developed a worldwide web-accessible computer program dubbed SMALI for scoring matrix-assisted ligand identification for SH2 domains and other signaling modules. The current version of SMALI harbors 76 unique scoring matrices for SH2 domains derived from screening oriented peptide array libraries. These scoring matrices are used to search a protein database for short peptides preferred by an SH2 domain. An experimentally determined cut-off value is used to normalize an SMALI score, therefore allowing for direct comparison in peptide-binding potential for different SH2 domains. SMALI employs distinct scoring matrices from Scansite, a popular motif-scanning program. Moreover, SMALI contains built-in filters for phosphoproteins, Gene Ontology (GO) correlation and colocalization of subject and query proteins. Compared to Scansite, SMALI exhibited improved accuracy in identifying binding peptides for SH2 domains. Applying SMALI to a group of SH2 domains identified hundreds of interactions that overlap significantly with known networks mediated by the corresponding SH2 proteins, suggesting SMALI is a useful tool for facile identification of signaling networks mediated by modular domains that recognize short linear peptide motifs.
Experts in peptide:MHC binding studies are often able to estimate the impact of a single residue substitution based on a heuristic understanding of amino acid similarity in an experimental context. Our aim is to quantify this measure of similarity to improve peptide:MHC binding prediction methods. This should help compensate for holes and bias in the sequence space coverage of existing peptide binding datasets.
Here, a novel amino acid similarity matrix (PMBEC) is directly derived from the binding affinity data of combinatorial peptide mixtures. Like BLOSUM62, this matrix captures well-known physicochemical properties of amino acid residues. However, PMBEC differs markedly from existing matrices in cases where residue substitution involves a reversal of electrostatic charge. To demonstrate its usefulness, we have developed a new peptide:MHC class I binding prediction method, using the matrix as a Bayesian prior. We show that the new method can compensate for missing information on specific residues in the training data. We also carried out a large-scale benchmark, and its results indicate that prediction performance of the new method is comparable to that of the best neural network based approaches for peptide:MHC class I binding.
A novel amino acid similarity matrix has been derived for peptide:MHC binding interactions. One prominent feature of the matrix is that it disfavors substitution of residues with opposite charges. Given that the matrix was derived from experimentally determined peptide:MHC binding affinity measurements, this feature is likely shared by all peptide:protein interactions. In addition, we have demonstrated the usefulness of the matrix as a Bayesian prior in an improved scoring-matrix based peptide:MHC class I prediction method. A software implementation of the method is available at: http://www.mhc-pathway.net/smmpmbec.
A monoclonal antibody (MAb) directed against an unknown Chlamydophila pneumoniae epitope has been characterized, and the respective peptide mimotope has been identified. A murine MAb specific for C. pneumoniae was used to select peptides from phage display libraries. The peptides identified from the phage display library clones reacted specifically with the respective target murine MAb and with human sera previously identified as having antibody titers to C. pneumoniae. The selected peptide mimotope sequences tended to be composed of charged residues surrounding a core of hydrophobic residues. The peptide with the best binding could inhibit >95% of binding to the MAb, suggesting that the selected peptide binds the paratope of the respective MAb. The peptide reacted with human sera previously determined by microimmunofluorescence to have anti-C. pneumoniae antibodies. The peptide was competitively competed with the MAb against Renografin-purified, sonicated C. pneumoniae in an enzyme-linked immunosorbent assay and with whole-cell C. pneumoniae in an indirect fluorescence assay format, demonstrating its potential utility in the development of diagnostics. The use of this novel peptide may allow investigators to establish standardized assays free from cross-reactive Chlamydia trachomatis and Chlamydophila psittaci epitopes and immunoreactivity.
The ability to screen compounds in a high-throughput manner is essential in the process of small molecule drug discovery. Critical to the success of screening strategies is the proper design of the assay, often implying a compromise between ease/speed and a biologically relevant setting. Leishmaniasis is a major neglected disease with limited therapeutic options. In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect–infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage. Screening of a 909-member library of bioactive compounds against Leishmania donovani revealed 59 hits in the promastigote primary screen and 27 in the intracellular amastigote screen, with 26 hits shared by both screens. This suggested that screening against the promastigote stage, although more suitable for automation, fails to identify all active compounds and leads to numerous false positive hits. Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite. This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy.
Leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a poverty-related disease threatening 350 million people throughout the world. Drugs currently available to treat this disease are toxic to the patient and drug-resistant parasites are emerging. New therapeutics are therefore needed. Fortunately, interest in confronting the treatment challenges has grown and new technology has led to an increase in high-throughput screens conducted against Leishmania. In order to gain insight into the most efficient screening strategy, we compared two methods, one targeting the easily cultured insect-infective promastigote stage of the parasite, and the other, targeting the clinically relevant but more difficult to culture intracellular amastigote stage. We show that while a screen against promastigotes is amenable to automation, it fails to recognize all active compounds. These compounds revealed only by an intracellular assay might act on host cell pathways important for parasite development. Targeting such pathways is an emerging strategy in drug discovery against infectious diseases.
Cryptococcus neoformans is surrounded by a capsular polysaccharide that inhibits phagocytosis of the yeast by macrophages. This capsular polysaccharide also confers several physicochemical properties to the cell surface, including a negative surface charge and a hydrophilic surface. The present study was designed to determine whether a hydrophobic surface was necessary or sufficient for phagocytosis of C. neoformans cells. The hydrophobic nature of the cell surface was measured by hydrophobic interaction chromatography on octyl-Sepharose. Liability to phagocytosis was determined by use of mouse peritoneal macrophages. The surface properties of C. neoformans cells were modified by (i) preincubation of cryptococcal cells with nonimmune serum or immune anticapsular serum, (ii) chemical modification of the carboxyl and O-acetyl groups in the capsular polysaccharide, and (iii) use of various serotypes of C. neoformans with different degrees of O-acetyl and xylosyl substitution. The results showed that it was possible to experimentally vary the surface hydrophobic-hydrophilic characteristics of the cell surface; however, the antiphagocytic character of the capsule remained unchanged. The results further suggest that a hydrophobic surface was neither necessary nor sufficient for phagocytosis of C. neoformans cells by macrophages.
Peptides or proteins near surfaces exhibit different structural properties from those present in a homogenous solution, and these differences give rise to varied biological activity. Therefore, understanding the detailed molecular structure of these molecules tethered to a surface is important for interpreting the performance of the various microarrays based on the activities of the immobilized peptides or proteins. We performed molecular dynamics simulations of a pentapeptide, RHSVV, an epitope of the tumor suppressor protein p53, tethered via a spacer on a functionalized silica surface and free in solution, to study their structural and conformational differences. These calculations allowed analyses of the peptide-surface interactions, the sequence-orientations and the translational motions of the peptide on surface to be performed. Conformational similarities are found among dominant structures of the tethered and free peptide. In the peptide microarray simulations, the peptide fluctuates between a parallel and tilted orientation driven in part by the hydrophobic interactions between the nonpolar peptide residues and the methyl-terminated silica surface. The perpendicular movement of the peptide relative to the surface is also restricted due to the hydrophobic nature of the microarray surface. With regard to structures available for recognition and binding, we find that similar conformations to those found in solution are available to the peptide tethered to the surface, but with a shifted equilibrium constant. Comparisons with experimental results show important implications of this for peptide microarray design and assays.
For genomic applications, signature-finding algorithms identify over-represented signatures (words) in collections of DNA sequences. The results can be presented as a specific sequence of bases, a consensus sequence showing possible combination of bases, or a matrix of weighted possibilities at each position. These results are often compared to a biological set of binding sites (i.e., known functional elements), which are usually represented as weighted matrices. The comparison is made by scoring the signatures against each weight matrix to identify the best option for a positive hit. However, this approach can misclassify results when applied to short sequences, which are a frequent result of signature finders. We describe a novel method using a window around the original sequences (those which the signature is based upon) to improve the comparison and identify a more significant measure of similarity. In doing so, our method transforms a list of DNA signatures into a resource of characterized binding sites with known functional roles and identifies novel elements in need of further elucidation.
position weight matrices; transcription factor binding sites; plane-sweep
A promising way in diagnostic and therapeutic applications is the development of peptide amphiphiles (PAs). Peptides with a palmitic acid alkylchain were designed and characterized to study the effect of the structure modifications on self-assembling capabilities and the multiple binding capacity to hemagglutinin (HA), the surface protein of influenza virus type A. The peptide amphiphiles consists of a hydrophilic headgroup with a biological functionality of the peptide sequence and a chemically conjugated hydrophobic tail. In solution they self-assemble easily to micelles with a hydrophobic core surrounded by a closely packed peptide-shell.
In this study the effect of a multiple peptide binding partner to the receptor binding site of HA could be determined with surface plasmon resonance measurements. The applied modification of the peptides causes signal amplification in relationship to the unmodified peptide wherein the high constant specificity persists. The molecular assembly of the peptides was characterized by the determination of critical micelle concentration (CMC) with concentration of 10-5 M and the colloidal size distribution.
The modification of the physico-chemical parameters by producing peptide amphiphiles form monomeric structures which enhances the binding affinity and allows a better examination of the interaction with the virus surface protein hemagglutinin.
CMC; Influenza virus detection; Micelle; PAs; Surface plasmon resonance
A semi-automated computational procedure to assist in the identification of bound ligands from unknown electron density has been developed. The atomic surface surrounding the density blob is compared to a library of three-dimensional ligand binding surfaces extracted from the Protein Data Bank (PDB). Ligands corresponding to surfaces which share physicochemical texture and geometric shape similarities are considered for assignment. The method is benchmarked against a set of well represented ligands from the PDB, in which we show that we can identify the correct ligand based on the corresponding binding surface. Finally, we apply the method during model building and refinement stages from structural genomics targets in which unknown density blobs were discovered. A semi-automated computational method is described which aims to assist crystallographers with assigning the identity of a ligand corresponding to unknown electron density. Using shape and physicochemical similarity assessments between the protein surface surrounding the density and a database of known ligand binding surfaces, a plausible list of candidate ligands are identified for consideration. The method is validated against highly observed ligands from the Protein Data Bank and results are shown from its use in a high-throughput structural genomics pipeline.
Electron density assignment; Function annotation; Ligand identification; Ligand assignment; Protein surfaces
Peptide aptamers are small proteins containing a randomized peptide sequence embedded into a stable protein scaffold, such as Thioredoxin. We developed a robust method for building a Combinatorial Library of Improved Peptide aptamers (CLIPs) of high complexity, containing ≥3×1010 independent clones, to be used as a molecular tool in the study of biological pathways. The Thioredoxin scaffold was modified to increase solubility and eliminate aggregation of the peptide aptamers. The CLIPs was used in a yeast two-hybrid screen to identify peptide aptamers that bind to various domains of the Receptor for Advanced Glycation End products (RAGE). NMR spectroscopy was used to identify interaction surfaces between the peptide aptamers and RAGE domains. Cellular functional assays revealed that in addition to directly interfering with known binding sites, peptide aptamer binding distal to ligand sites also inhibits RAGE ligand-induced signal transduction. This finding underscores the potential of using CLIPs to select allosteric inhibitors of biological targets.
Increasing evidence suggests that the effect of HLA-E on Natural Killer (NK) cell activity can be affected by the nature of the peptides bound to this non-classical, MHC class Ib molecule. However, its reduced cell surface expression, and until recently, the lack of specific monoclonal antibodies hinder studying the peptide-binding specificity HLA-E.
An in vitro refolding system was used to assess binding of recombinant HLA-E to either specific peptides or a nonamer random peptide library. Peptides eluted from HLA-E molecules refolded around the nonamer library were then used to determine a binding motif for HLA-E. Hydrophobic and non-charged amino acids were found to predominate along the peptide motif, with a leucine anchor at P9, but surprisingly there was no methionine preference at P2, as suggested by previous studies.
Compared to the results obtained with rat classical class Ia MHC molecules, RT1-A1c and RT1-Au, HLA-E appears to refold around a random peptide library to reduced but detectable levels, suggesting that this molecule's specificity is tight but probably not as exquisite as has been previously suggested. This, and a previous report that it can associate with synthetic peptides carrying a viral sequence, suggests that HLA-E, similar to its mouse counterpart (Qa-1b), could possibly bind peptides different from MHC class I leader peptides and present them to T lymphocytes.
Self-assembling peptides and peptide derivatives bearing cell-binding ligands are increasingly being investigated as defined cell culture matrices and as scaffolds for regenerative medicine. In order to systematically refine such scaffolds to elicit specific desired cell behaviors, ligand display should ideally be achieved without inadvertently altering other physicochemical properties such as viscoelasticity. Moreover, for in vivo applications, self-assembled biomaterials must exhibit low immunogenicity. In the present study, multi-peptide co-assembling hydrogels based on the beta-sheet fibrillizing peptide Q11 (QQKFQFQFEQQ) were designed such that they presented RGDS or IKVAV ligands on their fibril surfaces. In co-assemblies of the ligand-bearing peptides with Q11, ligand incorporation levels capable of influencing the attachment, spreading, morphology, and growth of human umbilical vein endothelial cells (HUVECs) did not significantly alter the materials’ fibrillization, beta-turn secondary structure, or stiffness. RGDS-Q11 specifically increased HUVEC attachment, spreading, and growth when co-assembled into Q11 gels, whereas IKVAV-Q11 exerted a more subtle influence on attachment and morphology. Additionally, Q11 and RGDS-Q11 were minimally immunogenic in mice, making Q11-based biomaterials attractive candidates for further investigation as defined, modular extracellular matrices for applications in vitro and in vivo.
Hydrocarbon stapling of peptides is a powerful technique to transform linear peptides into cell-permeable helical structures that can bind to specific biological targets. In this study, we have used high resolution solution NMR techniques complemented by Dynamic Light Scattering to characterize extensively a family of hydrocarbon stapled peptides with known inhibitory activity against HIV-1 capsid assembly to evaluate the various factors that modulate activity. The helical peptides share a common binding motif but differ in charge, the length and position of the staple. An important outcome of the study was to show the peptides share a propensity to self-associate into organized polymeric structures mediated predominantly by hydrophobic interactions between the olefinic chain and the aromatic side-chains from the peptide. We have also investigated in detail the structural significance of the length and position of the staple, and of olefinic bond isomerization in stabilizing the helical conformation of the peptides as potential factors driving polymerization. This study presents the numerous challenges of designing biologically active stapled peptides and the conclusions have broad implications for optimizing a promising new class of compounds in drug discovery.
The use of peptide libraries for the identification and characterization of T cell antigen peptide epitopes and mimotopes has been hampered by the need to form complexes between the peptides and an appropriate MHC molecule in order to construct a complete T cell ligand. We have developed a baculovirus-based peptide library method in which the sequence encoding the peptide is embedded within the genes for the MHC molecule in the viral DNA, such that insect cells infected with virus encoding a library of different peptides each displays a unique peptide–MHC complex on its surface. We have fished in such a library with two different fluorescent soluble T cell receptors (TCRs), one highly peptide specific and the other broadly allo-MHC specific and hypothesized to be much less focused on the peptide portion of the ligand. A single peptide sequence was selected by the former αβTCR that, not unexpectedly, was highly related to the immunizing peptide. As hypothesized, the other αβTCR selected a large family of peptides, related only by a similarity to the immunizing peptide at the p5 position. These findings have implications for the relative importance of peptide and MHC in TCR ligand recognition. This display method has broad applications in T cell epitope identification and manipulation and should be useful in general in studying interactions between complex proteins.
A baculovirus expression library -- encoding peptides in the context of MHC molecules -- has been developed to identify and characterize unique peptide-MHC complexes that bind specific T cell receptors
Histone and protein acetylation catalyzed by p300/CBP transcriptional coactivator regulates a variety of key biological pathways. This study investigates the proposed Theorell-Chance or “hit-and-run” catalytic mechanism of p300/CBP histone acetyltransferase (HAT) using bisubstrate analogs. A range of histone peptide tail peptide-CoA conjugates with different length linkers were synthesized and evaluated as inhibitors of p300 HAT. We show that longer linkers between the histone tail peptide and the CoA substrate moieties appear to allow for dual engagement of the two binding surfaces. Results with D1625R/D1628R double mutant p300 HAT further confirm the requirement for a negatively charged surface on the enzyme to interact with the histone tail.
Selective S1P4 receptor antagonists could be novel therapeutic agents for the treatment of influenza infection in addition to serving as a useful tool for understanding S1P4 receptor biological functions. 5-(2,5-dichlorophenyl)-N-(2,6-dimethylphenyl)furan-2-carboxamide was identified from screening the Molecular Libraries-Small Molecule Repository (MLSMR) collection and selected as a promising S1P4 antagonist hit with moderate in vitro potency and high selectivity against the other family receptor subtypes (S1P1–3,5). Rational chemical modifications of the hit allowed the disclosure of the first reported highly selective S1P4 antagonists with low nanomolar activity and adequate physicochemical properties suitable for further lead-optimization studies.
S1P4 receptor antagonists; S1P1–3,5 receptor family; 5-aryl furan-2-arylcarboxamide
We recently described ten peptides selected from a 16,384-member combinatorial library based on their ability to permeabilize synthetic lipid vesicles in vitro (Rathinakumar R and Wimley WC, J. Am. Chem. Soc. 2008, 130, 9849-9858). These peptides did not share a common sequence motif, length or net charge; nonetheless they shared a mechanism of action that is similar to the natural membrane permeabilizing antimicrobial peptides (AMP). To characterize the selected peptides and to compare the activity of AMPs in vivo and in vitro we report on the biological activity of the same selected peptides in bacteria, fungi, and mammalian cells. Each of the peptides has sterilizing activity against all classes of microbes tested, at 2-8 μM peptide, with only slight hemolytic or cytotoxicity against mammalian cells. Similar to many natural AMPs, bacteria are killed within a few minutes of peptide addition and the lethal step in vivo is membrane permeabilization. Single D-amino acid substitutions eliminated or diminished the secondary structure of the peptides and yet they retained activity against some microbes. Thus, secondary structure and biological activity are not coupled, consistent with the hypothesis that AMPs do not form pores of well defined structure in membranes, but rather destabilize membranes by partitioning into membrane interfaces and disturbing the organization of the lipids, a property that we have called “interfacial activity”. The observation that broad-spectrum activity, but not all antimicrobial activity, is lost by small changes to the peptides suggests that the in vitro screen is specifically selecting for the rare peptides that have broad-spectrum activity. We put forth the hypothesis that methods focusing on screening peptide libraries in vitro for members with the appropriate interfacial activity can enable the design, selection and discovery of novel, potent and broad-spectrum membrane-active antibiotics.
Collagens, characterized by a unique triple-helical structure, are the predominant component of extracellular matrices (ECMs) existing in all multicellular animals. Collagens not only maintain structural integrity of tissues and organs, but also regulate a number of biological events, including cell attachment, migration and differentiation, tissue regeneration and animal development. The specific functions of collagens are generally triggered by specific interactions of collagen-binding molecules (membrane receptors, soluble factors and other ECM components) with certain structures displayed on the collagen triple helices. Thus, synthetic triple-helical peptides that mimic the structure of native collagens have been used to investigate the individual collagen–protein interactions, as well as collagen structure and stability. The first part of this article illustrates the design of various collagen-mimetic peptides and their recent applications in matrix biology. Collagen is also acknowledged as one of the most promising biomaterials in regenerative medicine and tissue engineering. However, the use of animal-derived collagens in human could put the recipients at risks of pathogen transmission or allergic reactions. Hence, the production of safe artificial collagen surrogates is currently of considerable interest. The latter part of this article reviews recent attempts to develop artificial collagens as novel biomaterials.
biomaterial; collagen; extracellular matrix; peptide; supramolecule; triple helix
More and more disordered regions have been discovered in protein sequences, and many of them are found to be functionally significant. Previous studies reveal that disordered regions of a protein can be predicted by its primary structure, the amino acid sequence. One observation that has been widely accepted is that ordered regions usually have compositional bias toward hydrophobic amino acids, and disordered regions are toward charged amino acids. Recent studies further show that employing evolutionary information such as position specific scoring matrices (PSSMs) improves the prediction accuracy of protein disorder. As more and more machine learning techniques have been introduced to protein disorder detection, extracting more useful features with biological insights attracts more attention.
This paper first studies the effect of a condensed position specific scoring matrix with respect to physicochemical properties (PSSMP) on the prediction accuracy, where the PSSMP is derived by merging several amino acid columns of a PSSM belonging to a certain property into a single column. Next, we decompose each conventional physicochemical property of amino acids into two disjoint groups which have a propensity for order and disorder respectively, and show by experiments that some of the new properties perform better than their parent properties in predicting protein disorder. In order to get an effective and compact feature set on this problem, we propose a hybrid feature selection method that inherits the efficiency of uni-variant analysis and the effectiveness of the stepwise feature selection that explores combinations of multiple features. The experimental results show that the selected feature set improves the performance of a classifier built with Radial Basis Function Networks (RBFN) in comparison with the feature set constructed with PSSMs or PSSMPs that adopt simply the conventional physicochemical properties.
Distinguishing disordered regions from ordered regions in protein sequences facilitates the exploration of protein structures and functions. Results based on independent testing data reveal that the proposed predicting model DisPSSMP performs the best among several of the existing packages doing similar tasks, without either under-predicting or over-predicting the disordered regions. Furthermore, the selected properties are demonstrated to be useful in finding discriminating patterns for order/disorder classification.
A novel, high-throughput method for determining deacetylase substrate specificity was developed using a one-bead, one-compound (OBOC) acetyl-peptide library with a quantum dot tagging strategy and automated bead-sorting. A 5-mer OBOC peptide library of 104,907 unique sequences was constructed around a central ε-amino acetylated lysine. The library was screened using the human NAD+-dependent deacetylase SIRT1 for the most efficiently deacetylated peptide sequences. Beads preferentially deacetylated by SIRT1 were biotinylated and labeled with streptavidin-coated quantum dots. After fluorescent bead-sorting, the top thirty-nine brightest beads were sequenced by mass spectrometry. In-solution deacetylase assays on randomly chosen hit and non-hit sequences revealed that hits correlated with increased catalytic activity by as much as 20-fold. We found that SIRT1 can discriminate peptide substrates in a context dependent fashion.
Sir2; sirtuin; NAD; peptide library; one-bead; one-compound; deacetylase; quantum dot
A major hurdle in the application of therapeutic peptides is their rapid degradation by peptidases. Thioether bridges effectively protect therapeutic peptides against breakdown, thereby strongly increasing bioavailability, enabling oral and pulmonary delivery and potentially significantly optimizing the receptor interaction of selected variants. To efficiently select optimal variants, a library of DNA-coupled thioether-bridged peptides is highly desirable. Here, we present a unique cell surface display system of thioether-bridged peptides and successfully demonstrate highly selective screening. Peptides are posttranslationally modified by thioether bridge-installing enzymes in Lactococcus lactis, followed by export and sortase-mediated covalent coupling to the lactococcal cell wall. This allows the combinatorial optimization and selection of medically and economically highly important therapeutic peptides with strongly enhanced therapeutic potential.