Guanidyl-carboxylate interactions: crystal structures of arginine dipeptides*

The crystal structures of the hydrated dipeptides l -arginyl-l -aspartic acid and l -arginyl-l -glutamic acid have been determined from three-dimensional X-ray diffraction data. Each peptide crystallizes as a double-zwitterion with both the main and side-chain carboxyl groups ionized and the amino and guanidyl termini protonated. The peptide backbone conformation in both peptides is remarkably similar. Both peptides adopt a trans conformation for the peptide linkage with the guanidyl and acidic side-chains extended on opposite sides of the peptide backbone. The arginyl side-chain conformations differ between peptides; the conformation observed for arginyl aspartic acid is unique. Extensive intermolecular hydrogen bonding networks are observed in both structures; however, in neither structure is there evidence of intramolecular hydrogen bonding. The intermolecular guanidyl-carboxylate interactions are detailed. These interactions include a modified Type A interaction which models the possible bridging of adjacent peptide carbonyl oxygens in an α-helix by the guanidinium moiety.     

1H AND 13C NUCLEAR MAGNETIC RESONANCE SPECTRA OF SOME PEPTIDES WITH FIBRINOGEN-LIKE REACTIVITY

The ¹H and ¹³C spectra of four peptides, l -Phe-Val-Arg-pNA, d -Phe-Val-Arg-pNA, l -Phe-Pip-Arg-pNA and d -Phe-Pip-Arg-pNA (pNA = p-nitroaniline, Pip = pipecolic acid residue), have been examined, and deductions made about their conformation in solution. The d -Phe peptides, which are cleaved especially rapidly by thrombin in water, have structures (in deuterated DMSO) in which the aromatic ring of the d -Phe residue is folded back over the Val or Pip residue. This arrangement is not found in the l -Phe peptides. It is proposed that this feature (in which Phe could be situated near Val and near the Arg-Gly bond of the Aα chain in the three-dimensional structure of fibrinogen) may be specially advantageous for binding to the enzyme.     

Perspective: peptides as mimics of transmembrane segments in proteins

Abstract: Peptide-based approaches to protein structure within membranes have proven enormously valuable. When one focusses on the detailed manner through which membrane proteins actually traverse the cell bilayer, a simple observation emerges: helical peptide segments of 20 amino acids each constitute the only tangible connection between the inside and outside of the cell. Thus, a major step towards understanding the key relationships between biological function and membrane protein structure can be taken through characterization, by composition, sequence, chain length, hydrophobicity and conformation, of hydrophobic peptides designed as mimics of transmembrane segments.     

Conformational energy analysis of the chemotactic tripeptide formyl-Met-Leu-Phe and three analogs

Conformational energy analyses were carried out on the chemotactic tripeptide fMLF (CHO-Met-Leu-Phe) and three analogs fALF (CHO-Ala-Leu-Phe), fMF (CHO-Met-Phe), and MLF (H-Met-Leu-Phe). A near-folded or puckered conformation predominates in all four peptides. The calculated average end-to-end distance R of each of the peptides is 7.4 Å 7.6 Å, 7.0 Å, and 7.3 Å, respectively (where bends have R ≤ 7 Å and extended structures have R ? 10.5 Å). The puckered conformation calculated for fMLF is similar to that determined experimentally for fMLF in nonpolar solvents and in the protein receptor. The results suggest that maximum chemotactic activity of the peptides depends on a combination of the chemical structure (the presence of N-formyl-methionine) and backbone conformation (C, conformation of the first amino acid residue).     

Survey of conformational role of ester bonds in a cyclic depsipeptide

The effect of ester bond on the conformation of peptide molecule was studied by designing and synthesizing a model tetradepsipeptide cyclo(-l -Ala-l -Hmb-)z and by analyzing the conformation both theoretically and experimentally. Theoretical analysis showed that both ester and peptide bonds in the calculated low-energy conformations within 3 kcal/mol of the global minimum take a trans but distorted configuration. The distortion is larger in ester bonds than in peptide bonds. Further, the four carbonyls project from one side of the plane of the cyclic backbone, whereas the side chains project from the other side. These results are consistent with the experimental results obtained by NMR measurement; first, the coupling constant deduced from ¹H-NMR species in DMSO-d₆ is consistent with the dihedral angles of the calculated low-energy conformations; second, results of NOE measurement can reproduce the calculated configuration of the carbonyls and side chains. From the consistency between theoretical and experimental results, it is concluded that this model tetradepsipeptide takes an all-trans backbone conformation in solution and this backbone conformation is stabilized by large distortion in the ester bond, which compensates the strain resulted from the 12-membered cyclic backbone structure consisting only of L-residues.     

A convenient method for determining cyclic peptide conformation from 1D 1H-NMR information

A rapid and convenient method for determining the backbone conformation of cyclic peptides results from the combination of ID ¹H NMR information and molecular modeling. Φ, Angle torsional constraints calculated from ³J_HN,Hx coupling constants are used to determine the position of multiple-welled potential energy penalty functions that are imposed on the force field used in the structure refinement (Amber* with GB/SA solvation model). Monte Carlo searches and minimizations lead to a collection of structures that are clustered by backbone similarity and then filtered according to hydrogen-bonding constraints determined by the chemical shift temperature dependencies of the amide protons. This approach was applied to five cyclic peptides whose structures had been determined previously using more extensive 2D NMR techniques, and the importance of the torsional. H-bonding, and solvation restraints were assessed. For the four peptides that adopt a predominant conformation, this method reproduced the reported structures closely; lack of convergence for the fifth structure reflected the multiple backbone conformations that this macrocycle adopts. © Munksgaard 1996.     

Addition and omission analogs of the 13-residue antibacterial and hemolytic peptide PKLLKTFLSKWIG: structural preferences,model membrane binding and biological activities

The consequences of selective addition or deletion of polar amino acids in a 13-residue antibacterial peptide PKLLKTFLSKWIG on structure, membrane binding and biological activities have been investigated. The variants generated are (a) S and T residues replaced by K, (b) S and T residues deleted individually and together, (c) introduction of two additional K and (d) deletion of L and L with T. In the aqueous environment all the peptides were unordered. In trifluoroethanol, the spectra of peptides belonging to groups (a-c) suggest distorted helical conformation. Peptides in group (d) appear to adopt β-sheet conformation. The peptides bind to zwitterionic and negatively charged lipid vesicles, although to different extents. With the exception of peptides in group (d), all the other peptides exhibited comparable antibacterial activity against Escherichia coli and Staphylococcus aureus. However, the changes made in the peptides in groups (a-c) resulted in reduction of hemolytic activity compared to the parent peptide. Extent of binding to lipid vesicles composed of phosphatidylcholine and cholesterol appears to correlate with hemolytic activity. It appears that polar and charged residues play a major role in modulating the biological activities of the 13-residue peptide PKLLKTFLSKWIG. The 11-residue peptide-like PKLLKFLKWIG has selective antibacterial activity. Thus, by judicious engineering it should be possible to generate short peptides with selective antibacterial activity.     

Influence of the hydrophilic face on the folding ability and stability of α‐helix bundles: relevance to the peptide catalytic activity

Although not the sole feature responsible, the packing of amino acid side chains in the interior of proteins is known to contribute to protein conformational specificity. While a number of amphipathic peptide sequences with optimized hydrophobic domains has been designed to fold into a desired aggregation state, the contribution of the amino acids located on the hydrophilic side of such peptides to the final packing has not been investigated thoroughly. A set of self‐aggregating 18‐mer peptides designed previously to adopt a high level of α‐helical conformation in benign buffer is used here to evaluate the effect of the nature of the amino acids located on the hydrophilic face on the packing of a four α‐helical bundle. These peptides differ from one another by only one to four amino acid mutations on the hydrophilic face of the helix and share the same hydrophobic core. The secondary and tertiary structures in the presence or absence of denaturants were determined by circular dichroism in the far‐ and near‐UV regions, fluorescence and nuclear magnetic resonance spectroscopy. Significant differences in folding ability, as well as chemical and thermal stabilities, were found between the peptides studied. In particular, surface salt bridges may form which would increase both the stability and extent of the tertiary structure of the peptides. The structural behavior of the peptides may be related to their ability to catalyze the decarboxylation of oxaloacetate, with peptides that have a well‐defined tertiary structure acting as true catalysts.     

N- and Calpha-methylation in biologically active peptides: synthesis, structural and functional aspects

Numerous backbone constraints can be used to develop pseudopeptides or pseudomimetics of biologically active peptides. Among those, N- and Calpha-methyl amino acids that can be incorporated by solid-phase peptide synthesis in a bioactive sequence represent important tools to restrict phi and psi angles of peptide backbone. This review will focus on the chemical syntheses of N- and Calpha-methyl amino acids, their effects on peptide conformation and structure, and their role on the peptide stability towards enzymatic degradation and on the biological activities of the resulting analogues.     

Inhibitors of peptidases: how they influence the biological activities of substance P, neurokinins, bradykinin and angiotensin in guinea pig, hamster and rat urinary bladders.

Neurokinins, bradykinin and angiotensins were tested in isolated urinary bladder of the guinea pig, the hamster and the rat, in the absence and in presence of a variety of peptidase inhibitors in order to establish if peptide degradation interferes with the bladder contractions elicited by the three types of peptides. Indeed, the effects of neurokinins, bradykinin and angiotensin I in the guinea pig bladder were significantly enhanced by captopril (4.6 x 10(-6) mol/l), chymostatin (1 mg/l), phosphoramidon (4.6 x 10(-6) mol/l) and thiorphan (1.0 x 10(-6) mol/l), while only captopril was found to potentiate the effects of the same peptides in the rat bladder. The four peptidase inhibitors, as well as bacitracin were found to modify the responses of the hamster urinary bladder to one or another or to all three groups of peptides and to DiMeC7. The present results suggest that the urinary bladders of various species have different types of active proteolytic enzymes: only the angiotensin-converting enzyme appears to be present in the rat bladder, while the same enzyme and possibly two additional endopeptidases interfere with the myotropic effects of neurokinins, kinins and angiotensins in the guinea pig and the hamster bladder.     

Immunological characterization of a non-toxic peptide conferring protection against the toxic fraction (AahG50) of the Androctonus australis hector venom.

KAaH1 and KAaH2 are non-toxic peptides, isolated from the venom of the Androctonus australis hector (Aah) scorpion. In a previous study, we showed these peptides to be the most abundant (approximately 10% each) in the toxic fraction (AahG50) of the Aah venom. KAaH1 and KAaH2 showed high sequence identities (approximately 60%) with birtoxin-like peptides, which likewise are the major peptidic components of Parabuthus transvaalicus scorpion venom. Here, we report the immunological characterization of KAaH1 and KAaH2. These peptides were found to be specifically recognized by polyclonal antibodies raised against AahII, the most toxic peptide of Aah venom, and represents the second antigenic group, including toxins from different scorpion species in the world. Moreover, KAaH1 partially inhibits AahII binding to its specific antibody, suggesting some common epitopes between these two peptides. The identification of possible key antigenic residues in KAaH1 was deduced from comparison of its 3-D model with the experimental structure of AahII. Two clusters of putative antigenically important residues were found at the exposed surface; one could be constituted of V3 and D53, the other of D10, T15 and Y16. Polyclonal antibodies raised against KAaH1 in mice were found to cross-react with both AahII and AahG50, and neutralizing 5LD(50)/ml of the toxic fraction. Mice vaccinated with KAaH1 were protected against a challenge of 2LD(50) of AahG50 fraction. All these data suggest that KAaH1 has clear advantages over the use of the whole or part of the venom. KAaH1 is not toxic and could produce sera-neutralizing scorpion toxins, not only from Aah venom, but also toxins of other venoms from Buthus, Leiurus, or Parabuthus scorpion species presenting antigenically related toxins.     

Aib‐based peptide backbone as scaffolds for helical peptide mimics

Abstract: Helical peptides that can intervene and disrupt therapeutically important protein–protein interactions are attractive drug targets. In order to develop a general strategy for developing such helical peptide mimics, we have studied the effect of incorporating α‐amino isobutyric acid (Aib), an amino acid with strong preference for helical backbone, as the sole helix promoter in designed peptides. Specifically, we focus on the hdm2–p53 interaction, which is central to development of many types of cancer. The peptide corresponding to the hdm2 interacting part of p53, helical in bound state but devoid of structure in solution, served as the starting point for peptide design that involved replacement of noninteracting residues by Aib. Incorporation of Aib, while preserving the interacting residues, led to significant increase in helical structure, particularly at the C‐terminal region as judged by nuclear magnetic resonance and circular dichroism. The interaction with hdm2 was also found to be enhanced. Most interestingly, trypsin cleavage was found to be retarded by several orders of magnitude. We conclude that incorporation of Aib is a feasible strategy to create peptide helical mimics with enhanced receptor binding and lower protease cleavage rate.     

Cyclic tetrapeptides with sequences related to HC toxin. Conformations and cation binding

Peptides with sequences related to HC toxin (cyclo(l Ala-d Ala-l -Aoe-d Pro)) can adopt a conformation locked by three γ turns. A “structure — spectroscopy characteristics” relationship is proposed. These peptides can complex Mg++ cations and the binding is accompanied by a transconformation of the peptide backbone. The relevance with the biological activity of the toxin is discussed.     

Conformational study of a peptide epitope shows large preferences for β-turn conformations

The conformational preferences of the 7-residue peptide Glu-Val-Val-Pro-His-Lys-Lys was investigated using a global search algorithm, namely the Electrostatically Driven Monte Carlo (EDMC) method, and the ECEPP/2 potential energy function. This particular sequence corresponds to the N-terminal portion of a 19-residue peptide antigen whose three dimensional structure, when complexed to a cognate antibody, was reported recently. As a result of this study a series of low-energy conformations were identified showing a common folding pattern with residues Val-3, Pro-4, His-5 and Lys-6 forming a β turn. A comparison of the computed conformations with the one determined by X-ray crystallography in the antibody-antigen complex reveals marked similarities. In most of the cases rms deviations smaller than 1.1 Å were found for the backbone atoms of the four residues forming the turn. These results suggest that the recognition process is accomplished in this case through the interaction of the antibody with relatively stable conformers of the antigenic peptide.     

A CD strategy for the study of polypeptide folding/unfolding

The circular dichroism spectrum of the 20-residue immunogenic peptide from the foot-and-mouth disease virus (VP1; 141-160 of serotype A, subtype 12) was solvent- and temperature-dependent. Careful solvent titration revealed two isodichroic points and plateaux consistent with stepwise unfolding of specific stable conformations. Variable temperature studies in cryogenic solvents and urea perturbation were consistent with the existence of three conformational moieties, the left-handed extended helix, the a-helix, and the α-helix. The number of residues in each helix was confirmed by CD spectral simulations. The strategy described here can be used to determine the components of a conformational equilibrium and their statistical weights, to study peptide folding and unfolding and to determine the bioactive conformation(s) of linear peptides. The conclusions were supported by 2D-NMR studies. A new mechanism for the stabilization of left-handed extended helices and destabilization of a-helices by urea is proposed. The structure of the peptide as resolved by CD spectroscopy is of particular significance since the conformation of this antigenic sequence in situ has so far not been solved by X-ray crystallography.     

Conformational properties of trans Ac-Asn-Pro-Tyr-NHMe and trans Ac-Tyr-Pro-Asn-NHMe in dimethylsulfoxide and in water determined by multinuclear n.m.r. spectroscopy

Vicinal coupling constants between various nuclei provide backbone and side-chain conformational information for a series of asparagine- and tyrosine-containing peptides in DMSO and in H₂O. By enriching Tyr of Ac-Asn-Pro-Tyr-NHMe with ¹⁵N, it has been possible to distinguish between the resonances of the two side-chain β protons of Tyr. Analysis of the coupling constants in terms of the distributions of side-chain conformations in these peptides indicates that the addition of Asn to the Pro-Tyr sequence leads to a less random conformational distribution. When compared to the side-chain rotamer distribution of Ac-Asn-NHMe and Ac-Tyr-NHMe, particular Asn and Tyr side-chain conformations of Ac-Asn-Pro-Tyr-NHMe are stabilized in dimethylsulfoxide solution. The interaction(s) which stabilize a unique Tyr side-chain conformation of Ac-Asn-Pro-Tyr-NHMe in dimethylsulfoxide are not present in Ac-Ala-Pro-Tyr-NHMe and are unaffected by the addition of Val-Pro to the C-terminus of Asn-Pro-Tyr. In water, a preferential stabilization of one Asn side-chain conformation of Ac-Asn-Pro-Tyr-NHMe is also observed, while the Tyr side-chain rotamer distribution is similar to that of Ac-Tyr-NHMe. An interaction between the Asn side chain and the Pro-Tyr-NHMe backbone was previously shown to stabilize a β-bend conformation at Pro-Tyr in water. Data are also presented for Ac-Tyr-Pro-Asn-NHMe, for which local interactions do not stabilize particular backbone conformations in dimethylsulfoxide or in water. The conformations of the peptides studied here are relatively insensitive to temperatures between 27° and 62°, both in dimethylsulfoxide and in water. The sequences Asn-Pro-Tyr and Tyr-Pro-Asn occur in ribonuclease A, and these tripeptides serve as models for the interactions involved in the folding of this protein.     

Abbreviated nomenclature for cyclic and branched homo‐ and hetero‐detic peptides

Abstract: Amino acid sequences and linear or head‐to‐tail cyclopeptides can be represented conveniently in one‐line text formulae using the three‐letter symbols. However, other – but nonetheless important – topologies of peptides are ‘side chain‐to‐head (or tail)’, ‘backbone‐to‐backbone’, ‘side chain‐to‐side chain’ cyclopeptides, ‘side chain‐to‐side chain’ connected peptide strands, and branched peptides (like peptide dendrimers). In general, such structures cannot be described using the three‐letter symbols in one‐line text: a chemical structure editor is required for symbolic representations according to the IUPAC‐IUBMB recommendations. The aim of this contribution is to offer an unambiguous and general nomenclature system that enables researchers to represent all cyclic and branched homo‐ and hetero‐detic peptides in a coherent manner in one‐line text – as long as their as constituents can be represented in (three)‐letter codes. The application of this new nomenclature would overcome the existing difficulties and provide a way to express complex situations in the shortest way in order to highlight more clearly the salient points in a given scientific communication.     

Solution conformations of deltorphin-I obtained from combined use of quantitative 2D-NMR and energy calculations: A comparison with dermenkephalin

Deltorphin-I, Tyr-d -Ala-Phe-Asp-Val-Val-Gly-NH₂ and dermenkephalin, Tyr-d -Met-Phe-His-Leu-Met-Asp-NH₂, two highly related opioid peptides from frog skin, display very similar N-termini but strikingly different C-terminal tails. Nevertheless, both peptides are highly potent at, and exquisitely selective for the δ-opioid receptor. To identify common determinants concuring to the remarkably efficient targeting of deltorphin-I and dermenkephalin, combined use of quantitative two-dimensional nuclear magnetic resonance (53 dipolar interactions studied at four temperatures) and energy calculations using simulated annealing generated five groups of deltorphin-I conformers. These groups were pooled into two families whose overall conformation could be described either by a left-handed helix (Family I) or by a big loop (Family II), both stabilized by H-bonds. Proximity of D-Ala²-Phe³-Asp⁴ and Val⁵-Val⁶-Gly⁷ triads is an obvious structural similarity between almost all groups in both families of structures. Whereas differences between the two families originated mostly from a transition at Ψ Asp⁴ backbone dihedral angle, the backbone structures at segment 1–4 are similar and spatial arrangements of Tyr¹ (t) and Phe³ (g^–) are identical in one group of each family. Moreover, these two groups have a N-terminal tetrapeptide whose conformation most closely resembles that of a well-defined group of structures for dermenkephalin. Altogether, these results suggest that conformational attributes that are common to dermenkephalin and deltorphin-I, i.e., the backbone conformation of the N-terminal tetrapeptide and preferential orientations in the side-chain of Tyr¹ (t) and Phe3 (g^–) underlie their ability to bind with high selectivity to the δ-opioid receptor.     

Circular dichroism studies of relaxin and insulin peptide chains

The circular dichroism (CD) spectra of pig relaxin and pig insulin are similar, reflecting the known structural similarities between the two hormones. However, the conformational characteristics of the separate chains of insulin and relaxin show significant differences. The S-sulfo forms of insulin A and B chains and S-sulfo relaxin A chain have CD spectra consistent with largely unordered structures whereas the S-sulfo form of relaxin B-chain has at least 90%β-structure. This β-structure may explain the unusual solubility and adsorptive properties of the relaxin B-chain and the poor combination yields with A-chain. The relaxin B-chain changes to a largely unordered conformation if the peptide is shortened at the carboxyl terminus by six amino acid residues. This conformational change has important implications in planning relaxin synthesis strategy. Significant interactions and conformational changes are observed between the oxidized forms of the A and B chains of both relaxin and insulin. In using CD to monitor chain recombination of native relaxin peptides it was found that the spectra obtained differ depending on whether the reduced chains are separated or not separated from the reaction mixture prior to reoxidation. Although the spectra differ the biological activity was 20% in both cases. The remainder of the reoxidized but inactive material contains β-structures which make a greater contribution to the CD spectrum when the chains have been separated and processed than when they are reoxidized in situ.     

Immunochemistry of scorpion toxins Synthesis and antigenic properties of a model of a loop region specific to α-toxins

A region of the toxin II of the scorpion Androctonus australis Hector, possessing a loop structure, is shown to be antigenic. Some clear hints for the probable antigenic character of this region were obtained by the protruding properties of the loop region, as assessed by accessibility computations using atomic coordinates of the toxin and Lee-Richards algorithm. A synthetic replica of the loop region was obtained in a linear and cyclised form. Within the total anti-toxin antibody population, we have found and isolated those that recognize the model peptides. A high affinity binding of these specific antibodies to the parent toxin was demonstrated, affording experimental evidence for the antigenic properties of the loop region.