Nmr study of poly(aspartic acid). II. α- and β-Peptide bonds in poly(aspartic acid) prepared by common methods

The nature of peptide bonds in poly(aspartic acid) prepared by debenzylation of poly(β-benzyl-L -aspartate) under various conditions has been studied by means of nmr spectroscopy. It was established that the majority of the polymers prepared, as well as the commercially obtained polymer, contained aspartic acid linked in both α- and β-peptide bonds. The purest polymer, having practically undetectable amounts of β-bond, was prepared by debenzylation by HBr in trifluoroacetic acid. It was established that the β-bonds are formed via succinimides.     

Nmr study of poly(aspartic acid). I. α- and β-Peptide bonds in poly(aspartic acid) prepared by thermal polycondensation

The structure of poly(aspartic acid) prepared by thermal polycondensation has been studied by means of nmr spectroscopy. The analysis of the ¹³C-nmr spectra of the polymer at various pH values and comparison with the spectrum of poly(α-L -aspartic acid) revealed that the polymer contained aspartic acid linked in α- and β-peptide bonds. The mole fraction of the β-peptide bonds has been found to be 0.8 ± 0.1. The significance of the results for the evolutionary theory of S. W. Fox is mentioned.     

Design of Peptides Using α,β-Dehydro-residues: Synthesis,Crystal Structure and Molecular Conformation of N-Boc-L-Val-ΔPhe–ΔPhe-L-Ala-OCH3

To obtain general rules of peptide design using α,β-dehydro-residues, a sequence with two consecutive ΔPhe-residues, Boc-L -Val-ΔPhe–ΔPhe- L -Ala-OCH₃, was synthesized by azlactone method in solution phase. The peptide was crystallized from its solution in an acetone/water mixture (70:30) in space group P6₁ with a=b=14.912(3) Å, c= 25.548(5) Å, V=4912.0(6) ų. The structure was determined by direct methods and refined by a full matrix least-squares procedure to an R value of 0.079 for 2891 observed [I?3σ(I)] reflections. The backbone torsion angles ?₁=?54(1)°, ψ₁= 129(1)°, ω₁=?177(1)°, ?₂ =57(1)°, ψ₂=15(1)°, ω₂ =?170(1)°, ?₃=80(1)°, ψ₃ =7(2)°, ω₃=?177(1)°, ?₄ =?108(1)° and ψ^T₄=?34 (1)° suggest that the peptide adopts a folded conformation with two overlapping β-turns of types II and III′. These turns are stabilized by two intramolecular hydrogen bonds between the CO of the Boc group and the NH of ΔPhe³ and the CO of Val¹ and the NH of Ala⁴. The torsion angles of ΔPhe² and ΔPhe³ side chains are similar and indicate that the two ΔPhe residues are essentially planar. The folded molecules form head-to- tail intermolecular hydrogen bonds giving rise to continuous helical columns which run parallel to the c-axis. This structure established the formation of two β-turns of types II and III′ respectively for sequences containing two consecutive ΔPhe residues at (i+2) and (i+3) positions with a branched β-carbon residue at one end of the tetrapeptide.     

Cu(II)–poly(L-arginine) complexes. Potentiometric and spectral characterization of amine and peptide nitrogen ligands

The study of Cu(II)–poly(L -arginine) complexes by potentiometric titration, as well as by optical, circular dichroism, and infrared spectra, provides information about the nature of ligands and the coordination sphere around the metal ion. Three different complexes have been identified. The first, which is formed below pH 8, contains two guanidinium nitrogens and two water molecules at the corners of the coordination square. The constant of the overall process as determined by the Gregor method equals 2.0 ± 0.1 × 10^?9. The two other complexes form between pH 8 and 10.5 and they contain two guanidinium and two peptide nitrogens as nearest ligands. One of them is a monomer and the other probably a dimer, which differ in the symmetry of the coordination sphere around the cupric ion. The optical spectra of the three complexes show an absorption band at 260 nm that we have assigned to a charge-transfer transition between a σ metal nitrogen (amine) molecular orbital and a d_x2_?y2 metal orbital. The spectra of the two complexes containing peptide nitrogens exhibit another absorption band at 320 nm, which we have assigned to a charge transfer from a π orbital of the amide group to the d_x2_?y2 metal orbital.     

13C nuclear magnetic resonance study of the cis-trans isomerism in X-Pro-Pro tripeptides.

13C nuclear magnetic resonance has been used to characterize quantitatively the cis-trans isomerism about both peptide bonds in the tripeptides Ser-Pro-Pro and Arg-Pro-Pro. Detailed pH titration data indicate that the configuration about both peptide bonds is closely linked to titration of the terminal carboxyl group and, to a lesser extent, to titration of the terminal amino group. The Pro2 C-3 resonance has been found particularly useful for interpretation due to its sensitivity to the isomerization about both peptide bonds. Analysis of the probabilities of the trans-trans, cic-cis, cis-trans, and trans-cis isomers in aqueous solution indicates a stability decrease in the order given. Similarities in the isomerization behavior of the two peptides indicate that side chain interactions involving the first residue have very little effect on the observed cis/trans ratios. The sensitivity of the cis/trans ratio to titration of the terminal amino group is most readily explained on the basis of an indirect effect on carbonyl-carbonyl repulsion.     

Structural studies of poly(α-aminoisobutyric acid)

The electron-diffraction pattern of an oriented film of poly(α-aminoisobutyric acid) in the 3₁₀-helical conformation has been analyzed. The conformation was obtained by a linked-atom least-squares refinement of average values from crystal structures. Specimens treated with dichloracetic acid, to improve their crystallinity, conform to space group R3c with a = 21.8 Å, c = 5.95 Å. The structure contains channels that can accommodate molecules of dichloracetic acid. One molecule of acid per six residues fills the channels, and the R-factor then is 34% using 23 reflections. Ir evidence is presented to show that the acid may hydrogen bond to the peptide groups. Some reflections occasionally observed on the diffraction photographs are attributed to a 15/4 α-helix. The significance of the results is considered in relation to Aib-containing peptides.     

Self-assembly of trimethyl chitosan and poly(anionic amino acid)-peptide antigen conjugate to produce a potent self-adjuvanting nanovaccine delivery system

Short peptides derived from virulent pathogen proteins are promising antigens for the development of vaccines against infectious diseases. However, in order to mimic the danger signals associated with natural infection and stimulate an adaptive immune response, peptide antigens must be co-delivered with immune adjuvants. In this study, a group A streptococcus (GAS) M-protein derived B-cell epitope: J8, and universal T-helper epitope P25 containing peptides, were chemically coupled with different anionic amino acid-based polymers. The poly(anionic amino acid)-peptide antigen conjugates were mixed with trimethyl chitosan (TMC) to produce self-adjuvanting nanoparticulate vaccine candidates. TMC from two different sources were used to analyse their effect on immunogenicity. The nanoparticles produced from a peptide modified with 10 residues of polyglutamic acid and fungal TMC (NP5) stimulated production of the highest levels of serum antibodies in outbred mice. These antibodies were opsonic against all clinical GAS isolates tested.     

Free energies and equilibria of peptide bond hydrolysis and formation

For every n amino acids linked in a protein there are n − 1 peptide bonds. The free energy of peptide bond hydrolysis and formation in aqueous solution defines the equilibrium position between peptide and amino acid hydrolysis products. Yet few experimental values exist. With a minimum of assumptions, this paper deduces the free energies of hydrolysis of a variety of peptide bonds. Formation of a dipeptide from two amino acids is about eight times more difficult than subsequent condensations of an amino acid to a dipeptide or longer chain. Condensation of an amino acid to a peptide of any size is five times more difficult than joining two smaller peptides of at least dipeptide size. Thus in an abiogenesis scenario there is a kind of nucleation in peptide bond formation with the initial condensation of two amino acids to yield a dipeptide more difficult than subsequent condensations to a growing chain. © 1998 John Wiley & Sons, Inc. Biopoly 45: 351–353, 1998     

Conformational study of poly(α,β-L-aspartic acid)

The conformation of several samples of poly(α,β-L -Asp) with a molar fraction of β-bonds ranging from 0.1 to 0.55 was investigated by means of ir and CD spectroscopy and potentiometric titration and compared with the results obtained previously with poly(α-L -Asp). All samples investigated underwent a conformational change induced by changes in their degree of ionization: unpronounced ir absorption of amide V at 650 cm^?1 was shifted to 620 cm^?1 and substantially increased on deionization; CD spectra changed with the degree of ionization, passing through an isosbestic point; and the pattern of the titration curves was more complex than that of a simple polyelectrolyte. The conformation developing with the decreasing degree of ionization may be considered to be α-helix, as deduced according to the analogous behavior of other polypeptides. The extent of the conformational change in the individual samples depends on the molar fraction of β-bonds: the higher it is, the lower is the helix-forming ability of the sample.     

Design,synthesis, and conformation of a model peptide of endothelin with cystine-stabilized α-helix motif

A model 16-peptide of endothelin-1 (MET-1), which has the minimized sequence homology to the corresponding pan of endothelin-1 (ET-1), was designed to confirm the cystine-stabilized α-helix motif. The model structure consists of an extended structure, a β-turn part, and an α-helix structure that is stabilized by two disulfide bonds. The α-helix segment was designed to emphasize the amphiphilic nature. In order to combine the extended structure and the α-helix segment, a D -Ala-Pro sequence was selected to fix the β-turn. The model endothelin 16-peptide amide was synthesized by solid-phase synthesis on a 4-methylbenzhydrylamine resin. Its conformation was examined by CD and two-dimensional (2D) ¹H-nmr measurements. MET-1 showed similar CD patterns to ET-1 in both buffer and 50% aqueous trifluoroethanol solution. The 2D nmr experiments in 50% aqueous ethylene glycol revealed that MET-1 closely resembles the conformation of ET-1 with an extended structure, an α-helix, and a β-turn unit in the same position of the sequence. Furthermore, model peptides without disulfide bond(s) could not assume a stable structure in aqueous solution, while they did have similar α-helical content in 50% trifluoroethanol with MET-1. When the two disulfide bridges were simultaneously formed, the peptide with the correct disulfide bonds (MET-1) was obtained in threefold excess to the isomer (apamin type. MET-2). These findings obtained by the modeling of ET-1 showed an important role for the stabilization of peptide conformation with disulfide bonds. © 1994 John Wiley & Sons, Inc.     

Isodichroic point and the β–random coil transition of poly(S-carboxymethyl-L-cysteine) and poly(S-carboxyethyl-L-cysteine) in the absence of added salt

The β-coil transition of poly(S-carboxymethyl-L -cysteine) (poly[Cys(CH₂CO₂H)]) and poly(S-carboxyethyl-L -cysteine) (poly[Cys((CH₂)₂CO₂H)]) was followed by CD, potentiometric titration, and viscosity in the absence of added salt. These different properties give consistent results for poly[Cys((CH₂)₂CO₂H)]. The CD spectra of poly[Cys(CH₂CO₂H)] change considerably with the degree of neutralization α even for a low-molecular-weight sample incapable of forming the β-structure. Because of the superposition of this additional effect, the dependence of CD on α is inconsistent with titration data for the case of poly[Cys(CH₂CO₂H)], particularly when the nπ transition is used to follow the β-coil transition. The change of CD inherent to the β-coil transition is characterized by an isodichroic point: 215 nm for poly[Cys((CH₂)₂CO₂H)] and 218 nm for poly[Cys(CH₂CO₂H)]. A criterion supporting the stacking of the pleated sheet is suggested based on the isodichroic point.     

Conformational study of poly(α-L-aspartic acid)

The conformation of poly(α-L -aspartic acid) was investigated on a sample in which β-bonds were not detected. CD and ir spectroscopy showed that poly(α-L -aspartic acid) passes through a conformational change induced by changes of the degree of ionization that is accompanied by precipitation; the precipitate is probably highly helical. The change was also detected by potentiometric titration.     

Conformation-Determining role for the N-acetyl group in the O-glycosidic linkage, α-GalNAc-Thr

An ¹H-nmr study of 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-D-galactopyranose (AcGalNAc) glycosylated Thr-containing tripeptides in Me₂SO-d₆ solution reveals two mutually exclusive intramolecular hydrogen bonds. In Z-Thr(AcGalNAc)-Ala-Ala-OMe, there is an intramolecular hydrogen bond between the Thr amide proton and the sugar N-acetyl carbonyl oxygen. The strength of this hydrogen bond will be dependent on the amino acid residues on the Thr C terminal side to some undetermined distance. In Ac-Thr(AcGalNAc)-Ala-Ala-OMe, a different intramolecular hydrogen bond between the sugar N-acetyl amide proton and the Thr carbonyl oxygen exists. The choice of hydrogen bonds seems dependent on the bulkiness of the residues on the Thr N terminal side. The consequence of such strong hydrogen bonds is a clearly defined orientation of the sugar moiety with respect to the peptide backbone. In the former, the plane of the sugar pyranose ring is roughly oriented perpendicularly to the peptide backbone. The latter orientation is where the plane of the sugar ring is roughly in line with the peptide backbone. In both orientations, the sugar moiety can increase the shielding of the neighboring amino acid residues from the solvent. The idea that the amino acid residues near the glycosylated Thr influence orientation of the sugar moiety with respect to the peptide backbone and in turn possibly hinder peptide backbone flexibility has interesting implications in the conformational as well as the biological role of O-glycoproteins.     

Side-chain conformation in poly(γ-phenethyl-L-glutamate)

X-ray diffraction and energy-minimization results are reported for poly(γ-phenethyl-L -glutamate). Orthorhombic unit-cell parameters of drawn fibers are a = 15.4 Å, b = 26.6 Å, c = 54.4 Å. Atomic coordinates are derived for an α-helix peptide conformation that corresponds to a calculated side-chain internal energy minimum. The side-chain conformation correlates well with the electron density projection; the side chains wrap around the α-helical main chain with the phenethyl ester group directed toward the N-terminus. The para-axis of the benzene ring is inclined at an angle nearly nearly normal to the helix axis. The x-ray structure factors calculated for this model, when compared to the 10 observed structure factors, yield a crystallographic reliability index of R = 0.23.     

Diversity of Coding Sequences and Gene Structures of the Antifungal Peptide Mytimycin (MytM) from the Mediterranean Mussel, <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis>

Knowledge on antifungal biomolecules is limited compared to antibacterial peptides. A strictly antifungal peptide from the blue mussel, Mytilus edulis named mytimycin (MytM) was reported in 1996 as partial NH₂ 33 amino acid sequence. Using back-translations of the previous sequence, MytM-related nucleotide sequences were identified from a normalized Mytilus galloprovincialis expressed sequence tag library. Primers designed from a consensus sequence have been used to obtain a fragment of 560 nucleotides, including the complete coding sequence of 456 nucleotides. Precursor is constituted by a signal peptide of 23 amino acids, followed by MytM of 54 amino acids (6.2–6.3 kDa, 12 cysteines) and C-terminal extension of 75 amino acids. Only two major amino acid precursor sequences emerged, one shared by M. galloprovincialis from Venice and Vigo, the other belonging to M. galloprovincialis from Palavas, with nine amino acid differences between the two MytM. Predicted disulfide bonds suggested the presence of two constrained domains joined by amino acidic NIFG track. Intriguing was the presence of conserved canonical EF hand-motif located in the C-terminus extension of the precursor. The MytM gene was found interrupted by two introns. Intron 2 existed in two forms, a long (1,112 nucleotides) and a short (716 nucleotides) one resulting from the removal of the central part of the long one. Both the short (GenBank FJ804479) and the long (GenBank FJ804478) genes are simultaneously present in the mussel genome.     

Interaction of poly(I) and poly(I)·poly(C) with chlorodiethylenetriamino platinum(II) chloride

The fixation of dien-Pt on poly(I)·poly(C) leads to only minor changes in the uv and CD spectra at ambient temperature, showing that there is little perturbation of the secondary structure in the r_b range studied (up to 0.30). However, the melting profiles show two steps. The T_m for strand separation increases linearly from 61°C (r_b = 0) to 80°C (r_b = 0.18), after which it declines on further increasing the r_b. The second melting step is not complete at 100°C, and the magnitude of the absorbance change in this second step also appears to be at a maximum at r_b = 0.18. Although dien-Pt can only coordinate to one base, the nmr spectra at 80°C also show a second type of interaction with the adjacent bases, which is only destroyed in the presence of a strong denaturing agent, 5M guanidinium hydrochloride. From these results and the spectrophotometric data, we observe that dien-Pt forms a triple sandwich by hydrogen bonding of the platinum amino groups to the adjacent hypoxanthine bases (N⁷). The presence of these hydrogen bonds accounts for the increased stability (maximal at one Pt to three hypoxanthine bases) and their rupture is seen in the second melting step. No interaction has been observed with poly(C) strand. Reaction of dien-Pt with poly(I) shows the formation of the same triple sandwich structure in the nmr spectra.     

Peptide backbone cleavage by α‐amidation is enhanced at methionine residues

Cleavage reactions at backbone loci are one of the consequences of oxidation of proteins and peptides. During α‐amidation, the C_α–N bond in the backbone is cleaved under formation of an N‐terminal peptide amide and a C‐terminal keto acyl peptide. On the basis of earlier works, a facilitation of α‐amidation by the thioether group of adjacent methionine side chains was proposed. This reaction was characterized by using benzoyl methionine and benzoyl alanyl methionine as peptide models. The decomposition of benzoylated amino acids (benzoyl‐methionine, benzoyl‐alanine, and benzoyl‐methionine sulfoxide) to benzamide in the presence of different carbohydrate compounds (reducing sugars, Amadori products, and reductones) was studied during incubation for up to 48 h at 80 °C in acetate‐buffered solution (pH 6.0). Small amounts of benzamide (0.3–1.5 mol%) were formed in the presence of all sugars and from all benzoylated species. However, benzamide formation was strongly enhanced, when benzoyl methionine was incubated in the presence of reductones and Amadori compounds (3.5–4.2 mol%). The reaction was found to be intramolecular, because α‐amidation of a similar 4‐methylbenzoylated amino acid was not enhanced in the presence of benzoyl‐methionine and carbohydrate compounds. In the peptide benzoyl‐alanyl‐methionine, α‐amidation at the methionine residue is preferred over α‐amidation at the benzoyl peptide bond. We propose here a mechanism for the enhancement of α‐amidation at methionine residues. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Computationally designed β‐turn foldamers of γ‐peptides based on 2‐(aminomethyl)cyclohexanecarboxylic acid

The γ‐peptide β‐turn structures have been designed computationally by the combination of chirospecific γ 2 , 3 ‐residues of 2‐(aminomethyl)cyclohexanecarboxylic acid (γAmc₆) with a cyclohexyl constraint on the C^α?C^β bond using density functional methods in water. The chirospecific γAmc₆ dipeptide with the (2S,3S)‐(2R,3R) configurations forms a stable turn structure in water, resembling a type II′ turn of α‐peptides, which can be used as a β‐turn motif in β‐hairpins of Ala‐based α‐peptides. The γAmc₆ dipeptide with homochiral (2S,3S)‐(2S,3S) configurations but different cyclohexyl puckerings shows the capability to be incorporated into one of two β‐turn motifs of gramicidin S. The overall structure of this gramicidin S analogue is quite similar to the native gramicidin S with the same patterns and geometries of hydrogen bonds. Our calculated results and the recently observed results may imply the wider applicability of chirospecific γ‐peptides with a cyclohexyl constraint on the backbone to form various peptide foldamers. © 2012 Wiley Periodicals, Inc. Biopolymers 97:1018–1025, 2012.     

Prediction of ketoacyl synthase family using reduced amino acid alphabets

Ketoacyl synthases are enzymes involved in fatty acid synthesis and can be classified into five families based on primary sequence similarity. Different families have different catalytic mechanisms. Developing cost-effective computational models to identify the family of ketoacyl synthases will be helpful for enzyme engineering and in knowing individual enzymes’ catalytic mechanisms. In this work, a support vector machine-based method was developed to predict ketoacyl synthase family using the n-peptide composition of reduced amino acid alphabets. In jackknife cross-validation, the model based on the 2-peptide composition of a reduced amino acid alphabet of size 13 yielded the best overall accuracy of 96.44% with average accuracy of 93.36%, which is superior to other state-of-the-art methods. This result suggests that the information provided by n-peptide compositions of reduced amino acid alphabets provides efficient means for enzyme family classification and that the proposed model can be efficiently used for ketoacyl synthase family annotation.     

Chemical synthesis of the S‐linked glycopeptide,sublancin

Sublancin is an S‐linked glycopeptide produced by Bacillus subtilis 168 and consists of 37 amino acid residues with two disulfide bonds. In this study, we synthesized sublancin by Fmoc‐based solid‐phase peptide synthesis and chemoselective disulfide formation reactions. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.