Conformational study of palindromic tripeptides (GPG, IPI and KPK) in HIV-1 protease--a density functional theory study

A comparative study has been carried out on three palindromic tripeptides Gly-Pro-Gly, Ile-Pro-Ile and Lys-Pro-Lys which were present in HIV protein along with their analogues applying density functional computation at B3LYP/6-31G* level of theory. Discrepancy from the structural analysis has been noted for all the systems and it was found to be more for amide capped structure at the C terminal of proline. The puckering amplitude A and Phase angle P of the pyrrolidine ring of proline in the chosen palindromic tripeptides and their analogues were calculated from the endocyclic torsion angles. The minimum energy conformers lying well within the prescribed region of proline were obtained for the derived compounds from potential energy surface scan mentioning that no role has been played by its terminal residues. This is further supported by the simulated amide bands identifying the helical structure for all three palindromic tripeptides signifying the importance of proline. The molecular properties such as stabilization energy, chemical hardness along with dipole moment were calculated and interpreted. The values of Calpha-H(s) and the peptide backbone N-Calpha-CO for all the selected conformers specify the three palindromic tripeptides to have a symmetrical achiral structure.     

Conformational limitations of glycylsarcosine as a prototypic substrate for peptide transporters

Peptide transporters are present in all species to absorb the small peptides that occur ubiquitously as products of proteolysis. The broad substrate specificities of these systems allow them to be exploited therapeutically for delivery of peptidomimetic drugs in microbes and man. To this end, glycylsarcosine is currently used as a standard substrate for assaying peptidomimetic transport by peptide transporters. However, in this study we find it is unsuitable as a general substrate, based on assays of its transport by model bacterial peptide transporters and computer-based conformational analysis of its structure. Of the two generic transporters for di- and tripeptides, exemplified by Dpp and Tpp in Escherichia coli, only Dpp can transport glycylsarcosine. The explanation for this finding came from molecular modelling, which indicated that glycylsarcosine can adopt only a restricted range of conformers compared with typical dipeptides, and that of the conformers with a trans peptide bond, the majority have the specific psi and phi backbone torsion angles needed for molecular recognition and transport by Dpp but none possessed psi and phi torsions required for recognition by Tpp; moreover, 38% of its conformers have cis peptide bonds that are not substrates for any peptide transporter. Thus, using glycylsarcosine as substrate in competition assays with compounds that typically form conformers recognised by both types of peptide transporter will underestimate their transport. These findings have implications for assays of oral availability of peptidomimetic drugs such as beta-lactams, ACE inhibitors and anti-viral compounds, for which glycylsarcosine is routinely used.     

Quinine‐Based Zwitterionic Chiral Stationary Phase as a Complementary Tool for Peptide Analysis: Mobile Phase Effects on Enantio‐ and Stereoselectivity of Underivatized Oligopeptides

Peptide stereoisomer analysis is of importance for quality control of therapeutic peptides, the analysis of stereochemical integrity of bioactive peptides in food, and the elucidation of the stereochemistry of peptides from a natural chiral pool which often contains one or more D‐amino acid residues. In this work, a series of model peptide stereoisomers (enantiomers and diastereomers) were analyzed on a zwitterionic ion‐exchanger chiral stationary phase (Chiralpak ZWIX(+) 5 µm), in order to investigate the retention and separation performance for such compounds on this chiral stationary phase and elucidate its utility for this purpose. The goal of the study focused on 1) investigations of the effects of the sample matrix used to dissolve the peptide samples; 2) optimization of the mobile phase (enabling deriving information on factors of relevance for retention and separation); and 3) derivation of structure–selectivity relationships. It turned out that small di‐ and tripeptides can be well resolved under optimized conditions, typically with resolutions larger than 1.5. The optimized mobile phase often consisted of methanol–tetrahydrofuran–water (49:49:2; v/v/v) with 25 mM formic acid and 12.5 mM diethylamine. This work proposes some guidance on which mobile phases can be most efficiently used for peptide stereoisomer separations on Chiralpak ZWIX. Chirality 28:5–16, 2016. © 2015 Wiley Periodicals, Inc.     

Solid-phase precipitation and extraction, a new separation process applied to the isolation of synthetic peptides.

A new method for separation and purification is described. The process, referred to as solid-phase precipitation and extraction (SPPE), was developed and applied to postcleavage isolation of synthetic peptides. The technique uses normal approaches of chromatography and solid-phase extraction sorbents with a precipitation or drying procedure so that the sorbent becomes a support matrix for thin-film deposition of the compounds of interest. This procedure causes precipitated compounds of interest to be trapped on the large surface area or in the pores of the matrix so that by-products and impurities can be removed by strong wash solvents. In application to solid-phase peptide synthesis chemistry, by-products from the cleavage and deprotection are selectively extracted from the crude sample mixture under mild conditions. In comparison to the ether precipitation method used in peptide chemistry, the SPPE process provides isolated products that are 14-17% (w/w) higher purity.     

Quantitative measurements of vancomycin binding to self-assembled peptide monolayers on chips by quartz crystal microbalance

This paper describes direct binding of a small vancomycin to peptide ligands immobilized on a sensor chip using quartz crystal microbalance. In this study, the binding ligands were composed of three components: a molecular recognition element (peptide), a conformationally flexible and hydrophilic linker, and a long-chain alkanethiol. These peptide ligands were used to establish the well-packed, self-assembled monolayers on quartz chips and could be readily synthesized using conventional organic chemistry protocols. Results of quartz crystal microbalance measurements showed that vancomycin specifically associated with the d-Ala-d-Ala-containing peptide with an affinity of 3.2+/-0.3 microM and was, as expected, completely inactive to the self-assembled monolayer presenting l-Ala-l-Ala peptide. The dissociation constant obtained correlated well with values reported in literature and was further confirmed by surface plasmon resonance measurement (2.7+/-0.7 microM). The technique used in this study should be applicable to both peptidyl and nonpeptidyl ligands of greater complexity than that used here. This method is practical, it provides quantitative binding information, and complicated analysis is avoided.     

Mixtures of a series of homologous hydrophobic peptides with lipid bilayers: a simple model system for examining the protein-lipid interface

The interactions of several members of a homologous series of peptides with the phospholipid bilayer have been examined by using fluorescence and deuterium NMR spectroscopy, differential scanning calorimetry, and measurements of water-to-bilayer partition coefficients. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers and tripeptides of the form Ala-X-Ala-O-tert-butyl are used as a model system to probe the influence of amino acid side-chain substitution on the insertion of peptides into membranes and the behavior of peptide/bilayer mixtures. Tripeptides with X = Gly, Ala, Phe, and Trp have been examined. All of the tripeptides are water soluble, and all partition into DMPC bilayer vesicles to some extent. The Gly-containing peptide is the least soluble and the Trp-containing peptide the most soluble in the bilayer. The extent of perturbation of the bilayer structure induced by the peptides parallels their bilayer solubility: the Gly and Ala peptides act as simple impurities while peptides containing bulky aromatic rings cause a phase separation. Changes in the fluorescence properties of the Trp analogue upon incorporation into the bilayer indicate that the Trp side chain is probably immersed in the hydrocarbon region of the bilayer. Peptides of this form should serve as easily modifiable model systems with which to examine details of how the bilayer environment affects peptide conformation, as well as how hydrophobic peptides affect the bilayer structure.     

Hydrophobic interactions affect hydrogen bond strengths in complexes between peptides and vancomycin or ristocetin

A study on complexes of the glycopeptide antibiotics vancomycin and ristocetin with various dipeptides and tripeptides shows that the intermolecular hydrogen bond strengths of the complexes are reasonably well correlated with their free energy of formation. Such correlation is not anticipated on the basis of a purely hydrophobic interaction of the peptide side-chains with the antibiotics. It is also shown that the free energy changes observed are very different from those expected as a result of hydrophobic forces. These facts suggest that addition of a hydrophobic group not only allows hydrophobic bonding but also strengthens existing hydrogen bonds. The increased hydrogen bond strength can be an important factor in determining the overall binding energy.     

Examination of chiral stability during the preparation of hydrazides and coupling by the azide procedure using a series of model peptides

The peptide hydrazides of N-benzyloxycarbonylglycyl-X where X = alanine, leucine, phenylalanine, valine and isoleucine have been prepared by hydrazinolysis of the corresponding methyl esters and 2,4-distributed-5 (4H)-oxazolones, and by the carbodiimide-mediated reactions of the peptide acids with hydrazine in the presence of 1-hydroxybenzotriazole. The stereochemical purity of the products was examined by coupling them by the azide method with an ester of lysine followed by analysis for the diastereomeric tripeptides by an established procedure. The results concur with and substantiate the generally held notions on the chiral stability associated with hydrazinolysis and the azide procedure, except that an optically pure hydrazide could not be obtained from the oxazolone of the alanyl peptide.     

Specificity of peptide transport systems in Lactococcus lactis: evidence for a third system which transports hydrophobic di- and tripeptides.

A proton motive force-driven di-tripeptide carrier protein (DtpT) and an ATP-dependent oligopeptide transport system (Opp) have been described for Lactococcus lactis MG1363. Using genetically well-defined mutants in which dtpT and/or opp were inactivated, we have now established the presence of a third peptide transport system (DtpP) in L. lactis. The specificity of DtpP partially overlaps that of DtpT. DtpP transports preferentially di- and tripeptides that are composed of hydrophobic (branched-chain amino acid) residues, whereas DtpT has a higher specificity for more-hydrophilic and charged peptides. The toxic dipeptide L-phenylalanyl-beta-chloro-L-alanine has been used to select for a di-tripeptide transport-negative mutant with the delta dtpT strain as a genetic background. This mutant is unable to transport di- and tripeptides but still shows uptake of amino acids and oligopeptides. The DtpP system is induced in the presence of di- and tripeptides containing branched-chain amino acids. The use of ionophores and metabolic inhibitors suggests that, similar to Opp, DtpP-mediated peptide transport is driven by ATP or a related energy-rich phosphorylated intermediate.     

Quantitative analysis of the relationship between structure and antioxidant activity of tripeptides

Peptides from enzymatic hydrolysates of food proteins exhibit significant antioxidant activity. Several studies have attempted to determine the factors contributing to the antioxidant activity of peptides; however, the physicochemical properties and factors essential for the antioxidant activity of peptides are still unclear. In this study, in order to clarify the factors important for peptide antioxidant activity based on the properties of component amino acids, 55 tripeptides were synthesized from 20 natural amino acids and their antioxidant activity was measured using the Trolox equivalent antioxidant capacity (TEAC) assay system. The tripeptides were divided into two data sets: a training set comprising 50 compounds and a validated set comprising five compounds. The structure‐activity relationship of the training set was then analyzed using classical quantitative structure‐activity relationship (QSAR) analysis. The study findings demonstrate that the presence of a cysteine residue at any position, an aromatic amino acid at the C‐terminus, higher hydrophobicity of the N‐terminal residue, and smaller HOMO‐LUMO energy gap of the middle residue can significantly enhance the antioxidant activity. The activities of the five validated compounds were predicted using the constructed QSAR model, and a good correlation between measured and predicted activities was observed. The information obtained from the QSAR model could be useful for effective production of antioxidant peptides from food proteins such as egg white proteins.     

Screening of a Small Set of Random Peptides: A New Strategy to Identify Synthetic Peptides that Mimic Epitopes

Small diversity libraries, composed of 4550 synthetic dodecapeptides and 8000 synthetic tripeptides, have been used to identify sequences homologous to small linear and non-linear parts of epitopes. Here we report that synthetic peptides identified through alignment of dodecapeptides and tripeptides derived from these small libraries have, in direct ELISA and/or competitive ELISA, activities similar to that of peptides covering the native epitope and similar to that of peptides derived from large expression libraries composed of 10⁶–10⁷ random peptides. This result was obtained with the monoclonal antibodies 6A.A6 and M2. Mab 6A.A6 binds the transmissible gastroenteritis virus (TGEV) and mAb M2 binds the FLAG^®-peptide, an affinity tag. It was also found that the antibody binding activity of peptides, derived from small or large libraries, can strongly depend on the way in which the peptide is presented to the antibody, i.e. high antibody titers were obtained when these peptides were synthesized on pins or coated onto microtiter plates, whereas low IC₅₀s were obtained with these peptides in solution. We postulate that small peptide libraries may be a powerful tool to quickly identify new peptides that can be used as sensitive markers for mAbs of interest. © 1997 John Wiley & Sons, Ltd.     

A synthetic model of collagen: an experimental investigation of the triple-helix stability

Variations I–XVIII of a trimerlike cross-linked collagen model peptide were synthesized and used to investigate the cooperation of different neighboring Gly-X-Y tripeptides. The carboxy-terminal decapentapeptide of the tripe-helical part of collagen type I was chosen as the starting point of sequentially modified elongations. The transition temperatures determined by CD measurements show that the incorporation of the imino acid free tripeptides Gly-Ala-Ala and Gly-Ile-Ala results in a weakening of the triple-helical structure. It is demonstrated that the desired thermal stability of the collagen triple helix requires the “clustered” arrangement of helix-promoting tripeptides, especially of Gly-Pro-Hyp.     

Projection Structure of DtpD (YbgH), a Prokaryotic Member of the Peptide Transporter Family

Cellular uptake of di- and tripeptides has been characterized in numerous organisms, and various transporters have been identified. In contrast, structural information on peptide transporters is very sparse. Here, we have cloned, overexpressed, purified, and biochemically characterized DtpD (YbgH) from Escherichia coli, a prokaryotic member of the peptide transporter family. Its homologues in mammals, PEPT1 (SLC15A1) and PEPT2 (SLC15A2), not only transport peptides but also are of relevance for uptake of drugs as they accept a large spectrum of peptidomimetics such as β-lactam antibiotics, antivirals, peptidase inhibitors, and others as substrates. Uptake experiments indicated that DtpD functions as a canonical peptide transporter and is, therefore, a valid model for structural studies of this family of proteins. Blue native polyacrylamide gel electrophoresis, gel filtration, and transmission electron microscopy of single-DtpD particles suggest that the transporter exists in a monomeric form when solubilized in detergent. Two-dimensional crystallization of DtpD yielded first tubular crystals that allowed the determination of a projection structure at better than 19 Å resolution. This structure of DtpD represents the first structural view of a member of the peptide transporter family.     

Sensitivity to nikkomycin Z in Candida albicans: role of peptide permeases.

The uptake of tritiated nikkomycin Z, a potent inhibitor of chitin synthetase, is mediated by a peptide transport system in Candida albicans. Kinetic transport assays with radioactive di- and tripeptides and competition studies suggest that two distinct systems operate in this yeast. Nikkomycin Z was transported through one of these systems, common to di- and tripeptides. A peptide transport-deficient mutant was isolated on the basis of its resistance to nikkomycin Z. The mutant lost most of its capacity to take up dipeptides but simultaneously increased its ability to transport tripeptides. These results indicate that C. albicans handles peptides through multiple transport systems and adjusts their expression to environmental conditions.     

Angular variances for internal bond rotations of side chains in GXG-based tripeptides derived from (13)C-NMR relaxation measurements: Implications to protein folding

The study of backbone and side-chain internal motions in proteins and peptides is crucial to having a better understanding of protein/peptide "structure" and to characterizing unfolded and partially folded states of proteins and peptides. To achieve this, however, requires establishing a baseline for internal motions and motional restrictions for all residues in the fully, solvent-exposed "unfolded state." GXG-based tripeptides are the simpliest peptides where residue X is fully solvent exposed in the context of an actual peptide. In this study, a series of GXG-based tripeptides has been synthesized with X being varied to include all twenty common amino acid residues. Proton-coupled and -decoupled (13)C-nmr relaxation measurements have been performed on these twenty tripeptides and various motional models (Lipari-Szabo model free approach, rotational anisotropic diffusion, rotational fluctuations within a potential well, rotational jump model) have been used to analyze relaxation data for derivation of angular variances and motional correlation times for backbone and side-chain chi(1) and chi(2) bonds and methyl group rotations. At 298 K, backbone motional correlation times range from about 50 to 85 ps, whereas side-chain motional correlation times show a much broader spread from about 18 to 80 ps. Angular variances for backbone phi,psi bond rotations range from 11 degrees to 23 degrees and those for side chains vary from 5 degrees to 24 degrees for chi(1) bond rotations and from 5 degrees to 27 degrees for chi(2) bond rotations. Even in these peptide models of the "unfolded state," side-chain angular variances can be as restricted as those for backbone and beta-branched (valine, threonine, and isoleucine) and aromatic side chains display the most restricted motions probably due to steric hinderence with backbone atoms. Comparison with motional data on residues in partially folded, beta-sheet-forming peptides indicates that side-chain motions of at least hydrophobic residues are less restricted in the partially folded state, suggesting that an increase in side-chain conformational entropy may help drive early-stage protein folding. Copyright 1999 John Wiley & Sons, Inc.     

AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis

For the efficient translocation of organic nitrogen, small peptides of two to three amino acids are posited as an important alternative to amino acids. A new transporter mediating the uptake of di- and tripeptides was isolated from Arabidopsis thaliana by heterologous complementation of a peptide transport-deficient Saccharomyces cerevisiae mutant. AtPTR1 mediated growth of S. cerevisiae cells on different di- and tripeptides and caused sensitivity to the phytotoxin phaseolotoxin. The spectrum of substrates recognized by AtPTR1 was determined in Xenopus laevis oocytes injected with AtPTR1 cRNA under voltage clamp conditions. AtPTR1 not only recognized a broad spectrum of di- and tripeptides, but also substrates lacking a peptide bond. However, amino acids, omega-amino fatty acids or peptides with more than three amino acid residues did not interact with AtPTR1. At pH 5.5 AtPTR1 had an apparent lower affinity (K(0.5) = 416 microm) for Ala-Asp compared with Ala-Ala (K(0.5) = 54 microm) and Ala-Lys (K(0.5) = 112 microm). Transient expression of AtPTR1/GFP fusion proteins in tobacco protoplasts showed that AtPTR1 is localized at the plasma membrane. In addition, transgenic plants expressing the beta-glucuronidase (uidA) gene under control of the AtPTR1 promoter demonstrated expression in the vascular tissue throughout the plant, indicative of a role in long-distance transport of di- and tripeptides.     

Functional and structural characterization of a prokaryotic peptide transporter with features similar to mammalian PEPT1

The ydgR gene of Escherichia coli encodes a protein of the proton-dependent oligopeptide transporter (POT) family. We cloned YdgR and overexpressed the His-tagged fusion protein in E. coli BL21 cells. Bacterial growth inhibition in the presence of the toxic phosphonopeptide alafosfalin established YgdR functionality. Transport was abolished in the presence of the proton ionophore carbonyl cyanide p-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. YdgR transports selectively only di- and tripeptides and structurally related peptidomimetics (such as aminocephalosporins) with a substrate recognition pattern almost identical to the mammalian peptide transporter PEPT1. The YdgR protein was purified to homogeneity from E. coli membranes. Blue native-polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized YdgR suggest that it exists in monomeric form. Transmission electron microscopy revealed a crown-like structure with a diameter of approximately 8 nm and a central density. These are the first structural data obtained from a proton-dependent peptide transporter, and the YgdR protein seems an excellent model for studies on substrate and inhibitor interactions as well as on the molecular architecture of cell membrane peptide transporters.     

Aminopeptidase N from Streptococcus salivarius subsp. thermophilus NCDO 573: purification and properties

A 96 kDa aminopeptidase was purified from Streptococcus salivarius subsp. thermophilus NCDO 573. The enzyme had similar properties to aminopeptidases isolated from lactococci and lactobacilli and showed a high degree of N -terminal amino acid sequence homology to aminopeptidase N from Lactococcus lactis subsp. cremoris. It catalysed the hydrolysis of a range of aminoacyl 4-nitroanilides and 7-amido-4-methylcoumarin derivatives, dipeptides, tripeptides and oligopeptides. In common with aminopeptidases from other lactic acid bacteria, the enzyme from Strep. salivarius subsp. thermophilus showed highest activity with lysyl derivatives but was also very active with arginyl and leucyl derivatives. Relative activity with alanyl, phenylalanyl, tyrosyl, seryl and valyl derivatives was considerably lower and with glycyl, glutamyl and prolyl derivatives almost negligible. The aminopeptidase also catalysed the hydrolysis of dipeptides and tripeptides but mostly at rates much less than that with L-lysyl-4-nitroanilide and oligopeptides. The enzyme catalysed the successive hydrolysis of various amino acid residues from the N -terminus of several oligopeptides but it was unable to cleave peptide bonds on the N -terminal side of a proline residue.     

SH3-SPOT: an algorithm to predict preferred ligands to different members of the SH3 gene family

We have developed a procedure to predict the peptide binding specificity of an SH3 domain from its sequence. The procedure utilizes information extracted from position-specific contacts derived from six SH3/peptide or SH3/protein complexes of known structure. The framework of SH3/peptide contacts defined on the structure of the complexes is used to build a residue-residue interaction database derived from ligands obtained by panning peptide libraries displayed on filamentous phage.The SH3-specific interaction database is a multidimensional array containing frequencies of position-specific contacts. As input, SH3-SPOT requires the sequence of an SH3 domain and of a query decapeptide ligand. The array, that we call the SH3-specific matrix, is then used to evaluate the probability that the peptide would bind the given SH3 domain. This procedure is fast enough to be applied to the entire protein sequence database.Panning experiments were performed to search putative specific ligands of different SH3 domains in a database of decapeptides, or in a database of protein sequences. The procedure ranked some of the natural partners of interaction of a number of SH3 domains among the best ligands of the approximately 5. 6x10(9) different decapeptides in the SWISSPROT database. We expect the predictive power of the method to increase with the enrichment of the SH3-specific matrix by interaction data derived from new complex structures or from the characterization of new ligands. The procedure was developed using the SH3 domain family as test case but its application can easily be extended to other families of protein domains (such as, SH2, MHC, EH, PDZ, etc.).     

De Novo Design and Experimental Characterization of Ultrashort Self-Associating Peptides

Self-association is a common phenomenon in biology and one that can have positive and negative impacts, from the construction of the architectural cytoskeleton of cells to the formation of fibrils in amyloid diseases. Understanding the nature and mechanisms of self-association is important for modulating these systems and in creating biologically-inspired materials. Here, we present a two-stage de novo peptide design framework that can generate novel self-associating peptide systems. The first stage uses a simulated multimeric template structure as input into the optimization-based Sequence Selection to generate low potential energy sequences. The second stage is a computational validation procedure that calculates Fold Specificity and/or Approximate Association Affinity (K^*_association) based on metrics that we have devised for multimeric systems. This framework was applied to the design of self-associating tripeptides using the known self-associating tripeptide, Ac-IVD, as a structural template. Six computationally predicted tripeptides (Ac-LVE, Ac-YYD, Ac-LLE, Ac-YLD, Ac-MYD, Ac-VIE) were chosen for experimental validation in order to illustrate the self-association outcomes predicted by the three metrics. Self-association and electron microscopy studies revealed that Ac-LLE formed bead-like microstructures, Ac-LVE and Ac-YYD formed fibrillar aggregates, Ac-VIE and Ac-MYD formed hydrogels, and Ac-YLD crystallized under ambient conditions. An X-ray crystallographic study was carried out on a single crystal of Ac-YLD, which revealed that each molecule adopts a β-strand conformation that stack together to form parallel β-sheets. As an additional validation of the approach, the hydrogel-forming sequences of Ac-MYD and Ac-VIE were shuffled. The shuffled sequences were computationally predicted to have lower K^*_association values and were experimentally verified to not form hydrogels. This illustrates the robustness of the framework in predicting self-associating tripeptides. We expect that this enhanced multimeric de novo peptide design framework will find future application in creating novel self-associating peptides based on unnatural amino acids, and inhibitor peptides of detrimental self-aggregating biological proteins.