13C- and 1H-nmr evidence for all-cis peptide bonds in cyclic tetrapeptide cyclo(L-Pro-Sar)2

Conformations of the cyclic tetrapeptide cyclo(L -Pro-Sar)₂ in solution were studied by ¹H- and ¹³C-nmr spectrometry and model building. The nmr data provide definite evidence that this cyclic peptide exists chiefly in two conformations, namely, a C₂-symmetric conformation and an asymmetric structure. The former was demonstrated to be predominant in polar solvents (100% in Me₂SO-d₆). This structure contains all cis-peptide bond linkages and all trans′ Pro C^α?CO bonds. It represents the first cyclic tetrapeptide in which all four peptide bonds have been found in the cis-conformation. As the polarity of the solvent decreases, the population of C₂-symmetric conformers decreases (88% in CD₃CN and 65% in CDCl₃). At the same time, a minor asymmetric conformer, characterized by cis-cis-cis-trans peptide bond sequences (two cis Sar-Pro bonds, one cis Pro-Sar bond, and one trans Pro-Sar bond), is seen to increase (9% in CD₃CN and 30% in CDCl₃). A proposed predominant conformation in solution for cyclo(L -Pro-Sar)₂ was compared with a crystal structure, as reported in an accompanying paper. Both structures show striking overall similarities.     

A Novel Route to Pseudoproline (Ψ<Superscript>H,H</Superscript>Pro)-Containing Dipeptides Building Blocks

In view of the synthetic and biological interest of pseudoproline (Ψ^R,RPro)-containing peptides, we have investigated a new strategy to these target compounds, taking into account the existence of the well-known ring–chain tautomerism occurring in the NH-free pseudoproline unit. Indeed, the NH-free oxaprolines derived from β-hyroxyamino acid (Ser, Thr) are rarely isolable in contrast to the cysteine-derived thiaproline. The strategy developed herein is based on the use of 2:3 adducts (amino acid–formaldehyde) resulting from the condensation of amino acids (Ser, Thr, Cys) with paraformaldehyde. The latter may exist as an equilibrium mixture of 1,4-diaza-3,9-dioxabicyclo[4.4.1]undecane (2) and isomeric N,N-methylenebis (oxazolidine) or -(thiazolidine) (3). Coupling these 2:3 adducts with a C-activated amino acid using conventional procedure afforded C2-unsubstituted pseudoproline (Ψ^H,HPro)-containing dipeptides. This strategy was applied to both oxa- and thiaprolines. Such result clearly established the usefulness of these 2:3 adducts in peptide synthesis as they allow to trap the non isolable NH-free oxaprolines. The isomerization process 2 3 appeared to play a major role in this procedure, as illustrated by the peculiar case of the serine-derived 2:3 adduct 2a where no coupling occurred.     

Structure of an early native‐like intermediate of β2‐microglobulin amyloidogenesis

To investigate early intermediates of β2‐microglobulin (β2m) amyloidogenesis, we solved the structure of β2m containing the amyloidogenic Pro32Gly mutation by X‐ray crystallography. One nanobody (Nb24) that efficiently blocks fibril elongation was used as a chaperone to co‐crystallize the Pro32Gly β2m monomer under physiological conditions. The complex of P32G β2m with Nb24 reveals a trans peptide bond at position 32 of this amyloidogenic variant, whereas Pro32 adopts the cis conformation in the wild‐type monomer, indicating that the cis to trans isomerization at Pro32 plays a critical role in the early onset of β2m amyloid formation.     

The effect of phosphorylation on the conformation of oligo-peptides with Ser–Pro motif: a molecular dynamics simulation

Model tetrapeptide system was designed to investigate the cis/trans isomerization of peptidyl-prolyl imide bond of Ser–Pro motif. To establish the side-chain O-phosphorylation effect in regulating the peptides conformations, molecular dynamics (MD) simulations where carried out on the designed tetrapeptides and their corresponding phosphorylated forms by MD Insight II Discovery3 approach. The most stable configurations and the statistic cis/trans concentration distribution demonstrated that the phosphorylation evidently influences the peptidyl-prolyl imide bond isomerization and works as a key effect in regulating the peptide conformations. The charge state and the site provided for the charge of the phosphate moiety might be an important key. The results also demonstrated that phosphorylation changes the cis conformation ratio of the peptide and the maximum cis value is obtained when the phosphate group has no negative charge.     

Synthesis and conformation of the cyclic octapeptides cyclo(Phe-Pro)4, cyclo(Leu-Pro)4, and cyclo[Lys(Z)-Pro]4

Cyclic octapeptides, cyclo(X-Pro)₄, where X represents Phe, Leu, or Lys(Z), were synthesized and their conformations investigated. A C₂-symmetric conformer containing two cis peptide bonds was found in all of these cyclic octapeptides. The numbers of available conformations due to the cis–trans isomerization of Pro peptide bonds depended on the nature of the solvent and X residue: they decreased in the following order: cyclo[Lys(Z)-Pro]₄ > cyclo(Leu-Pro)₄ > cyclo(Phe-Pro)₄ in CDCl₃. ¹³C spin-lattice relaxation times (T₁) of these cyclic octapeptides were measured, and the contribution of segmental mobility to T₁ was found to vary with the nature of the X residue.     

Effect of the Phosphate Group with Different Negative Charges on the Conformation of Phosphorylated Ser/Thr-Pro Motif

Summary Ser/Thr-Pro motif is a widespread phosphorylated site in proteins, and its reversible phosphorylation is an important regulatory progress in many cell cycles and signal transduction. Recent research reveals that phosphorylation affects the local conformation of the peptide and its binding with the substrate through peptidyl--prolyl cis/trans isomerization. In order to further explore the effect of the phosphate group with different charges, four model peptides containing non- and phosphorylated Ser/Thr-Pro motif were synthesized using the classical solid-phase method. 1H-NMR, TOCSY, and ROESY were employed to characterize the conformation of the model peptides in solution with different pH value and analyze the peptidyl--prolyl isomerization at a molecular level. The results demonstrate that phosphorylation increases the cis conformation in the peptide and the maximum cis/trans ratio is obtained when the phosphate group has two negative charges. Furthermore, the experiments prove that the phosphorylation introduces a hydrogen bond between the phosphate and the NH of Ser/Thr residue, and the charges of the phosphate affect certain conformations of the phosphorylated Ser/Thr-Pro motif.     

Synthesis and diuretic activities of pseudoproline‐containing analogues of the insect kinin core pentapeptide

C‐2 dimethylated/unmethylated thiazolidine‐4‐carboxylic acid and C‐2 dimethylated oxazolidine‐4‐carboxylic acid were introduced into the insect kinin core pentapeptide in place of Pro³, yielding three new analogues. NMR analysis revealed that the peptide bond of Phe²‐pseudoproline (ΨPro)³ is practically 100% in cis conformation in the case of dimethylated pseudoproline‐containing analogues, about 50% cis for the thiazolidine‐4‐carboxylic acid analogue and about 33% cis for the parent Pro³ peptide. The diuretic activities are consistent with the population of cis conformation of the Phe²‐ΨPro³/Pro³ peptide bonds, and the results confirm a cis Phe‐Pro bond as bioactive conformation. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

<Emphasis Type="Italic">Cis</Emphasis>–<Emphasis Type="Italic">trans</Emphasis> photoisomerization properties of GFP chromophore analogs

The photoswitching behaviour of the green fluorescent protein (GFP) chromophore and its analogs opens up exciting horizons for the engineering and development of molecular devices for high sensitivity in vivo studies. In this work we present the synthesis and photophysical study of four GFP chromophore analogs belonging to butenolide and pyrrolinone classes. These chromophores possess an intriguing photoinduced cis–trans isomerization mechanism. Stereochemical structural assignment was unambiguously performed by 1D Nuclear Overhauser Effect NMR measurements. The spectroscopic properties of both cis and trans isomers were studied, and photoconversion quantum yield for cis–trans isomerization was assessed to be in the 0.1–0.4 range. Finally, the ³J_C,H coupling constant in the ¹³C–C=C–H motif was in excellent agreement with theoretical DFT calculations, thus providing a further confirmation of cis–trans photoisomerization of the structurally analog GFP chromophore.     

The rate-limiting step in the folding of the cis-Pro167Thr mutant of TEM-1 β-lactamase is the trans to cis isomerization of a non-proline peptide bond

The stability and kinetics of unfolding and refolding of the P167T mutant of the TEM-1 β-lactamase have been investigated as a function of guanidine hydrochloride concentration. The activity of the mutant enzyme was not significantly modified, which strongly suggests that the Glu166–Thr167 peptide bond, like the Glu166–Pro167, is cis. The mutation, however, led to a significant decrease in the stability of the native state relative to both the thermodynamically stable intermediate and the fully unfolded state of the protein. In contrast to the two slower phases seen in the refolding of the wild-type enzyme, only one phase was detected in the refolding of the mutant, indicating a determining role of proline 167 in the kinetics of folding of the wild-type enzyme. The former phases are replaced by rapid refolding when the enzyme is unfolded for short periods of time, but the latter is independent of the time of unfolding. The monophasic refolding reaction of the mutant is proposed to reflect mainly the trans→cis isomerization of the Glu166–Thr167 peptide bond. © 1996 John Wiley & Sons, Inc.     

Steric effects of cis-trans isomerism on neighboring residues in proline oligopeptides: A 13C-nmr study of conformational heterogeneity in linear tripeptides

A series of proline-containing linear oligopeptides (4 dipeptides and 15 tripeptides) were synthesized and examined in aqueous and nonaqueous solutions using ¹³C-nmr spectroscopy. Spectra of linear tripeptides showing cis-trans isomerism about the X-Pro bond (X = Pro, Gly, and Ala) also show neighboring effects on the chemical shifts of residues both preceding and following the prolyl moiety. The extent of cis-trans isomerism observed about the X-Pro peptide bond correlates not only with the nature of X, but also depends on the size of the residue following proline; the larger substituents favor an increase in cis content about the X-Pro bond.     

1H- and 13C-nmr investigations on cis–trans isomerization of proline peptide bonds and conformation of aromatic side chains in H-Trp-(Pro)n-Tyr-OH peptides

¹H and ¹³C high-resolution nmr spectra of cationic, zwitterionic, and anionic forms of the peptides: H-Trp-(Pro)_n-Tyr-OH, n = 0-5, and H-Trp-Pro-OCH₃ were obtained in D₂O solution. Analysis of H^α(Pro₁), H^α(Trp), C^γ(Pro), H^ε(Tyr), and H^δ(Trp) resonances provided evidence for the presence of two predominant backbone isomers: the all-trans one and another with the Trp-Pro peptide bond in cis conformation; the latter constituted about 0.8 molar fraction of the total peptide (n > 1) concentration. Relative content of these isomers varied in a characteristic way with the number of Pro residues and the ionization state of the peptides. The highest content of the cis (Trp-Pro) isomer, 0.74, was found in the anionic form of H-Trp-Pro-Tyr-OH; it decreased in the order of: anion ? zwitterion ≈ cation, and with the number of Pro residues to reach the value of 0.42 in the cationic form of H-Trp- (Pro)₅-Tyr-OH. Isomerization equilibria about Pro-Pro bond(s) were found to be shifted far (?0.9) in favor of the trans conformation. Interpretation of the measured vicinal coupling constants J_α?β′ and J_α?β″ for C^αH-C^βH₂ proton systems of Trp and Tyr side chains in terms of relative populations of g⁺, g^?, and t staggered rotamers around the χ₁ dihedral angle indicated that in all the peptides studied (a) rotation of Trp indole ring in cis (Trp-Pro) isomers is strongly restricted, and (b) rotation of Tyr phenol ring is relatively free. The most preferred χ₁ rotamer of Trp (0.8-0.9 molar fraction) was assigned as the t one on the basis of a large value of the vicinal coupling constant between the high-field H^β and carbonyl carbon atoms of Trp, estimated for the cis (Pro₁) form of H-Trp-Pro-Tyr-OH from a ¹H, ¹³C correlated spectroscopy ¹H detected multiple quantum experiment. This indicates that cis ? trans equilibrium in the Trp-Pro fragment is governed by nonbonding interactions between the pyrrolidine (Pro) and indole (Trp) rings. A molecular model of the terminal cis Trp-Pro dipeptide fragment is proposed, based on the presented nmr data and the results of our molecular mechanics modeling of low-energy conformers of the peptides, reported elsewhere. © 1993 John Wiley & Sons, Inc.     

Influence of lithium cations on prolyl peptide bonds

The influence of lithium cations on the cis/trans isomerization of prolyl peptide bonds was investigated in a quantitative manner in trifluoroethanol (TFE) and acetonitrile, employing NMR techniques. The focus was on various environmental and structural aspects, such as lithium cation and water concentrations, the type of the partner amino acid in the prolyl peptide bond, and the peptide sequence length. Comparison of the thermodynamic parameters of the isomerization in LiCl/TFE and TFE shows a lithium cation concentration dependence of the cis/trans ratio, which saturates at cation concentrations >200 mM. A pronounced increase in the cis isomer content in the presence of lithium cations occurs with the exception of peptides with Gly‐Pro and Asp‐Pro moieties. The cation effect appears already at the dipeptide level. The salt concentration can considerably be reduced in solvents with a lower number of nucleophilic centers like acetonitrile. The lithium cation effect decreases with small amounts of water and disappears at a water concentration of about 5%. The isomerization kinetics under the influence of lithium cations suggests a weak cation interaction with the carbonyl oxygen of the peptide bond. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

β‐Amino acids containing peptides and click‐cyclized peptide as β‐turn mimics: a comparative study with ‘conventional’ lactam‐ and disulfide‐bridged hexapeptides

The increasing interest in click chemistry and its use to stabilize turn structures led us to compare the propensity for β‐turn stabilization of different analogs designed as mimics of the β‐turn structure found in tendamistat. The β‐turn conformation of linear β‐amino acid‐containing peptides and triazole‐cyclized analogs were compared to ‘conventional’ lactam‐ and disulfide‐bridged hexapeptide analogs. Their 3D structures and their propensity to fold in β‐turns in solution, and for those not structured in solution in the presence of α‐amylase, were analyzed by NMR spectroscopy and by restrained molecular dynamics with energy minimization. The linear tetrapeptide Ac‐Ser‐Trp‐Arg‐Tyr‐NH₂ and both the amide bond‐cyclized, c[Pro‐Ser‐Trp‐Arg‐Tyr‐D ‐Ala] and the disulfide‐bridged, Ac‐c[Cys‐Ser‐Trp‐Arg‐Tyr‐Cys]‐NH₂ hexapeptides adopt dominantly in solution a β‐turn conformation closely related to the one observed in tendamistat. On the contrary, the β‐amino acid‐containing peptides such as Ac‐(R)‐β³‐hSer‐(S)‐Trp‐(S)‐β³‐hArg‐(S)‐β³‐hTyr‐NH₂, and the triazole cyclic peptide, c[Lys‐Ser‐Trp‐Arg‐Tyr‐βtA]‐NH₂, both specifically designed to mimic this β‐turn, do not adopt stable structures in solution and do not show any characteristics of β‐turn conformation. However, these unstructured peptides specifically interact in the active site of α‐amylase, as shown by TrNOESY and saturation transfer difference NMR experiments performed in the presence of the enzyme, and are displaced by acarbose, a specific α‐amylase inhibitor. Thus, in contrast to amide‐cyclized or disulfide‐bridged hexapeptides, β‐amino acid‐containing peptides and click‐cyclized peptides may not be regarded as β‐turn stabilizers, but can be considered as potential β‐turn inducers. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Investigating diproline segments in proteins: occurrences, conformation and classification

The covalent linkage between the side‐chain and the backbone nitrogen atom of proline leads to the formation of the five‐membered pyrrolidine ring and hence restriction of the backbone torsional angle ? to values of ?60 °± 30° for the L ‐proline. Diproline segments constitute a chain fragment with considerably reduced conformational choices. In the current study, the conformational states for the diproline segment ( ^L Pro‐ ^L Pro) found in proteins has been investigated with an emphasis on the cis and trans states for the Pro‐Pro peptide bond. The occurrence of diproline segments in turns and other secondary structures has been studied and compared to that of Xaa‐Pro‐Yaa segments in proteins which gives us a better understanding on the restriction imposed on other residues by the diproline segment and the single proline residue. The study indicates that P_II–P_II and P_II–α are the most favorable conformational states for the diproline segment. The analysis on Xaa‐Pro‐Yaa sequences reveals that the Xaa‐Pro peptide bond exists preferably as the trans conformer rather than the cis conformer. The present study may lead to a better understanding of the behavior of proline occurring in diproline segments which can facilitate various designed diproline‐based synthetic templates for biological and structural studies. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 54–64, 2012.     

Cation binding cyclic peptides composed of imino acid residues

Cyclo(L -Pro-Sar)_n (n = 2–4) with moderate flexibility and hydrophobicity of molecular structure was synthesized, and the characteristics of these cyclic peptides and their metal complexes in acetonitrile were investigated in connection with the residual properties using ¹³C-nmr measurements. The cyclic tetrapeptide cyclo(L -Pro-Sar)₂ showed a sterically hindered phenomenon in acetonitrile in which the amide backbone adopted a cis-trans-cis-trans sequence. The cyclic hexapeptide cyclo(L -Pro-Sar)₃ existed as a mixture of several conformers whose interconversion is slow on the nmr time scale, including cis-cis-trans and/or cis-trans-trans arrangement of the Sar-Pro bond. Finally, it was demonstrated that the cyclic octapeptide cyclo(L -Pro-Sar)₄ behaved as a mixture of multiple conformers which allowed for cis-trans isomerism about the Pro-Sar peptide bond, of which 20–30% had the all-cis Sar-Pro bond isomer and the remaining 70–80% had one (or more) cis Sar-Pro bond isomer. ¹³C-nmr spectra also demonstrated that cyclo(L -Pro-Sar)_n (n = 3,4) formed a 1:1 ion complex whose conformation was characterized by an all-trans peptide bond in the presence of excess metal salt. Cation binding studies, using CD measurements, established that the ion selectivity of cyclo(L -Pro-Sar)₄ in acetonitrile decreased in the order, Ba²⁺ > Ca²⁺ > Na⁺ > Mg²⁺ > Li⁺.     

Trypanosomatid Pin1‐Type Peptidyl‐Prolyl Isomerase Is Cytosolic and Not Essential for Cell Proliferation

Pin1‐type peptidyl‐prolyl cis/trans isomerases (PPIases) isomerise the peptide bond of specific phosphorylated (Ser/Thr)‐Pro residues, regulating various cellular events. Previously, we reported a Pin1‐type PPIase in Trypanosoma cruzi, but little is known about its function and subcellular localization. Immunofluorescence analysis revealed that in contrast with Pin1‐like proteins from diverse organisms, TcPin1 mainly localized in the cytoplasm and was excluded from the nuclei. In addition, RNAi‐mediated downregulation of TbPin1 in Trypanosoma brucei did not abolish cell proliferation. Using yeast two‐hybrid assay, we identified a MORN domain‐containing protein as putative Pin1‐binding partners. These data suggest that Pin1‐mediated signaling mechanism plays a different role in protozoan parasites.     

Potent and selective oxytocin receptor agonists without disulfide bridges

Oxytocin (OT) is a neuropeptide involved in a wide variety of physiological actions, both peripherally and centrally. Many human studies have revealed the potential of OT to treat autism spectrum disorders and schizophrenia. OT interacts with the OT receptor (OTR) as well as vasopressin 1a and 1b receptors (V_1aR, V_1bR) as an agonist, and agonistic activity for V_1aR and V_1bR may have a negative impact on the therapeutic effects of OTR agonism in the CNS. An OTR-selective agonistic peptide, FE 202767, in which the structural differences from OT are a sulfide bond instead of a disulfide bond, and N-alkylglycine replacement for Pro at position 7, was reported. However, the effects of amino acid substitutions in OT have not been comprehensively investigated to compare OTR, V_1aR, and V_1bR activities. This led us to obtain a new OTR-selective analog by comprehensive amino acid substitutions of OT and replacement of the disulfide bond. A systematic amino acid scanning (Ala, Leu, Phe, Ser, Glu, or Arg) of desamino OT (dOT) at positions 2, 3, 4, 5, 7, and 8 revealed the tolerability for the substitution at positions 7 and 8. Further detailed study showed that trans-4-hydroxyproline (trans-Hyp) at position 7 and γ-methylleucine [Leu(Me)] at position 8 were markedly effective for improving receptor selectivity without decreasing the potency at the OTR. Subsequently, a combination of these amino acid substitutions with the replacement of the disulfide bond of dOT analogs with a sulfide bond (carba analog) or an amide bond (lactam analog) yielded several promising analogs, including carba-1-[trans-Hyp⁷,Leu(Me)⁸]dOT (14) with a higher potency (7.2 pM) at OTR than that of OT and marked selectivity (>10,000-fold) over V_1aR and V_1bR. Hence, we investigated comprehensive modification of OT and obtained new OT analogs that exhibited high potency at OTR with marked selectivity. These OTR-selective agonists could be useful to investigate OTR-mediated effects on psychiatric disorders.     

Determination of metabolites of pyrethroids in human urine using solid-phase extraction and gas chromatography–mass spectrometry

The described method permits the determination of the five most important metabolites of the pyrethroids permethrin, cypermethrin, deltamethrin, λ-cyhalothrin, fenvalerate, phenothrin and β-cyfluthrin in human urine in one run. The major urinary metabolites of these substances are cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl₂CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl₂CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br₂CA), fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA). After acidic hydrolysis to release the conjugated carboxylic acid metabolites, the analytes were separated from the matrix by means of solid-phase extraction using a reversed-phase column. The components of the eluate were converted to their methyl esters and extracted in hexane. Separation and quantitative analysis of the pyrethroid metabolites was carried out by capillary gas chromatography and mass selective detection. 2-Phenoxybenzoic acid served as an internal standard. The detection limits lay between 0.3 and 0.5 μg per litre urine. The relative standard deviations of the within-series imprecision were between 1% and 6%. The relative recovery rates ranged between 90% and 98%. Using this method we determined the elimination of pyrethroid metabolites in 24-h urine samples from eight pest controllers after indoor application of permethrin. The detected concentrations ranged from 1 to 70 μg g⁻¹ creatinine.     

Multiple interconverting conformers of the cyclic tetrapeptide tentoxin, [cyclo-(L-MeAla1-L-Leu2-MePhe[(Z)Δ]3-Gly4)], as seen by two-dimensional 1H-nmr spectroscopy

The conformations of the phytotoxic cyclic tetrapeptide tentoxin [cyclo-(L -MeAla¹-L -Leu²-MePhe[(Z)Δ]³-Gly⁴ )] have been studied in aqueous solution by two-dimensional proton nmr at various temperatures. Contrary to what is observed in chloroform, tentoxin exhibits multiple exchanging conformations in water. Aggregation phenomena were also observed. Four conformations with different proportions (51, 37, 8, and 4%) were observed at ?5°C. Models were constructed from nmr parameters and restrained molecular dynamics simulations. All the models exhibit cis-trans-cis-trans conformation of the amide bond sequence. The conversion from one form to another is accomplished by a conformational peptide flip consisting of a 180° rotation of a nonmethylated peptide bond. © 1995 John Wiley & Sons, Inc.     

trans-Stilbene degradation by Arthrobacter sp. SL3 cells

trans-Stilbene degradation was examined by the reaction using resting cells of microorganisms isolated through the enrichment culture using trans-stilbene. The strain SL3, showing the highest trans-stilbene-degrading activity, was identified as Arthrobacter sp. One of the reaction products was identified to be cis,cis-muconic acid. Arthrobacter sp. SL3 cells also transformed benzaldehyde, benzoic acid and catechol into cis,cis-muconic acid, suggesting that one benzene ring of trans-stilbene was converted into cis,cis-muconic acid via benzaldehyde formed by its C_α=C_β bond cleavage.