Beta-turns induced in bradykinin by (S)-alpha-methylproline.

The ability of (S)-alpha-methylproline (alpha-MePro) to stabilise reverse-turn conformations in the peptide hormone bradykinin (BK = Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) has been investigated. Two BK analogues containing alpha-MePro at position 3 or position 7 were synthesised and their conformations in aqueous solution investigated by NMR spectroscopy. Whereas BK is largely disordered on the NMR time scale both analogues showed ROE connectivities in 2D-ROESY spectra indicative of reverse-turn conformations at both Pro2-Phe5 and Ser6-Arg9, whose formation appears to be cooperative. Some potential applications of alpha-MePro as a reverse-turn mimetic in the construction of synthetic peptide libraries is discussed.     

金铁锁根中的环肽成分

从云南民间重要的药用植物金铁锁（Ｐｓａｍｍｏｓｉｌｅｎｅ ｔｕｎｉｃｏｉｄｅｓ Ｗ．Ｃ．Ｗｕ ｅｔ Ｃ．Ｙ．Ｗｕ）的根中分离得到２个新的天然环二肽以及２个新的环八肽：金铁锁环肽Ａ和Ｂ（ｐｓａｍｍｏｓｉｌｅｎｉｎｓ Ａ ａｎｄ Ｂ）。它们的结构经光谱方法鉴定为ｃｙｃｌｏ（－Ａｌａ－Ａｌａ－）,ｃｙｃｌｏ（－Ｖａｌ－Ａｌａ－）,ｃｙｃｌｏ（－Ｐｒｏ１－Ｐｈｅ１－Ｐｒｏ２－Ｐｈｅ２－Ｐｈｅ３－Ａｌａ－ｐ     

Strategies for Positioning Fluorescent Probes and Crosslinkers on Formyl Peptide Ligands

Abstract

Chemoattractant receptors represent a major subset of the G-protein coupled receptor (GPCR) family. One of the best characterized, the N-formyl peptide receptor (FPR), participates in host defense responses of neutrophils. The features of the ligand which regulate its interaction with the FPR are well-known. By manipulating these features we have developed new ligands to probe structural and mechanistic aspects of the peptide-receptor interaction. Three ligand groups have been developed: 1) ligands containing a Lys residue located in positions 2 through 7 that can be conjugated to FITC (N-formyl-Met1-Lys2-Phe3-Phe4, N-formyl-Met1-Leu2-Lys3-Phe4, N-formyl-Met1-Leu2-Phe3-Lys4, N-formyl-Met1-Leu2-Phe3-Phe4-Lys5, N-formyl-nLeu1-Leu2-Phe3-nLeu4-Tyr5-Lys6 and N-formyl-Met1-Leu2-Phe3-Phe4-Gly5-Gly6-Lys7; 2) fluorescent pentapeptide ligands (N-formyl-Met-X-Phe-Phe-Lys(FITC) where X = Leu, Ala, Val or Gly); and 3) small crosslinking ligands where the photoaffinity crosslinker 4-azidosalicylic acid (ASA) was conjugated to Lys in positions 3 and 4 and p-benzoyl-phenylalanine (Bpa) was located in position 2 in N-formyl-Met1-Bpa2-Phe3-Tyr4. The peptides were characterized according to activity and affinity in human neutrophils and cell lines transfected with FPR. All of the peptides were agonists, with parallel affinity and activity. In the first group, the peptide activity decreases as Lys is placed closer to the N-formyl group and the activity is improved by 1–3 orders of magnitude by conjugation with FITC. In the second group, the dissociation rate of the peptide from the receptor increases as position 2 is replaced by aliphatic amino acids with smaller alkyl groups. In the third group, crosslinking ligands remain biologically active, display nM affinity and covalently label the FPR.     

A crystal molecular conformation of leucine-enkephalin related to the morphine molecule

Leucine-enkephalin (Try1-Gly2-Gly3-Phe4-Leu5) has been crystallized as a trihydrate from water solution. X-ray diffraction reveals a tightly folded molecular conformation with two fused beta III- (Gly2-Gly3) and beta I- (Gly3-Phe4) turns. The Tyr1 and Phe4 aromatic rings have a close orthogonal arrangement analogous to the tyramine and cyclohexenyl rings in morphine. This suggests that the conformation found in the trihydrate crystal structure could be required for recognition by mu-receptor sites.     

Peptide conformation. 48. Conformation and biological activity of proline containing cyclic retro-analogues of somatostatin

Six cyclic retro-analogues of the peptide hormone somatostatin have been synthesized using the solid phase technique. The peptides cyclo(-Xaa1-Phe2-Thr3-Lys4-Ybb5-Phe6-) and cyclo(-Phe1-Xaa2-Thr3-Lys4-Ybb5-Phe6-) with Xaa = D- or L-Pro and Ybb = D- or L-Trp were cyclized via the azide method. The conformations of the cyclic hexapeptides in DMSO-d6 solution were determined by a number of homo- and heteronuclear two-dimensional n.m.r.-techniques including 2D rotating frame NOE-spectroscopy. Two-step coherence transfers, ROE and chemical exchange, are observed for the first time in ROESY spectra. The backbone conformation of the all-trans cyclopeptides consists of a beta-turn containing the Pro residue in the position i + 1. These retro-analogues of somatostatin exhibit a high activity in the inhibition of cholate and phalloidin uptake by liver cells (cytoprotective effect); however, the hormonal activities of the natural hormone are completely suppressed. The constitutional and conformational requirements for the cytoprotective activity are discussed.     

Cyclopeptides from Sagina japonica (Caryophyllaceae)

Two new cyclic peptides, named sajaponicin C (1) and sajaponicin D (2), were isolated from the whole plants of Sagina japonica (Caryophyllaceae). Their structures were determined as cyclo(Pro(2)-Leu(2)-Tyr-Leu(1)-Phe(1)-Pro(3)-Phe(2)-Pro(1)) (1) and cyclo(Pro(1)-Pro(2)-Pro(3)-Pro(4)-Phe(1)-Gly-Thr-Ser-Phe(2)-Ile-Tyr) (2) on the basis of spectroscopic data, especially by two-dimensional (2D) NMR techniques.     

Enzymatic inactivation of bradykinin by rat brain neuronal perikarya

1. Bradykinin (Bk; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg8) inactivation by bulk isolated neurons from rat brain is described. 2. Bk is rapidly inactivated by neuronal perikarya (4.2 +/- 0.6 fmol/min/cell body). 3. Sites of inactivating cleavages, determined by a kininase bioassay combined with a time-course Bk-product analysis, were the Phe5-Ser6, Pro7-Phe8, Gly4-Phe5, and Pro3-Gly4 peptide bonds. The cleavage of the Phe5-Ser6 bond inactivated Bk at least five fold faster than the other observed cleavages. 4. Inactivating peptidases were identified by the effect of inhibitors on Bk-product formation. The Phe5-Ser6 bond cleavage is attributed mainly to a calcium-activated thiol-endopeptidase, a predominantly soluble enzyme which did not behave as a metalloenzyme upon dialysis and was strongly inhibited by N-[1(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate and endo-oligopeptidase A antiserum. Thus, neuronal perikarya thiol-endopeptidase seems to differ from endo-oligopeptidase A and endopeptidase 24.15. 5. Endopeptidase 24.11 cleaves Bk at the Gly4-Phe5 and, to a larger extent, at the Pro7-Phe8 bond. The latter bond is also cleaved by angiotensin-converting enzyme (ACE) and prolyl endopeptidase (PE). PE also hydrolyzes Bk at the Pro3-Gly4 bond. 6. Secondary processing of Bk inactivation products occurs by (1) a rapid cleavage of Ser6-Pro7-Phe8-Arg8 at the Pro7-Phe8 bond by endopeptidase 24.11, 3820ACE, and PE; (2) a bestatin-sensitive breakdown of Phe8-Arg9; and (3) conversion of Arg1-Pro7 to Arg1-Phe5, of Gly4-Arg9 to both Gly4-Pro7 and Ser6-Arg9, and of Phe5-Arg9 to Ser6-Arg9, Phe8-Arg9, and Ser6-Pro7, by unidentified peptidases. 7. A model for the enzymatic inactivation of bradykinin by rat brain neuronal perikarya is proposed.     

Synthesis and hydrolysis by pepsin and trypsin of a cyclic hexapeptide containing lysine and phenylalanine.

1. A cyclic hexapeptide, cyclo(-Gly2-Phe2-Gly-Lys-), and the corresponding open-chain hexapeptides, Gly2-Phe2-Gly-Lys and Phe-Gly-Lys-Gly2-Phe, have been synthesized and their susceptibilities to the hydrolytic action of pepsin and trypsin were determined. 2. The cyclic peptide was hydrolyzed slowly by trypsin to a hexapeptide Gly2-Phe2-Gly-Lys, the value of the Michaelis constant for this reaction being Km equals 0.00022 M. 3. The cyclic peptide was not cleaved by pepsin at all, but Gly2-Phe2-Gly-Lys was hydrolyzed rapidly at a Phe-Phe bond; Km equals 0.0091 M. 4. The cyclic peptide inhibits the hydrolysis of Gly2-Phe2-Gly-Lys by pepsin in a linear non-competitive manner, the value of the inhibition constant being Ki equals 0.004 M.     

Chiral resolution of phenylalanine by d-Phe imprinted membrane considering rejection property

An enantio-selective d-Phe imprinted P(AA-co-AN) membrane was prepared using the wet-phase inversion method. The membrane not only selectively adsorbed phenylalanine but also rejected it with a rejection selectivity of 0.82–0.64 and 0.91–0.63 during the filtration of 100 and 10 ppm (g m⁻³) racemate solutions, respectively. The fluxes of d-Phe and l-Phe during filtration of 10 ppm racemate solution were 0.0077–0.0229 and 0.0064–0.0208 mg m⁻² s⁻¹, respectively, and the fluxes of d-Phe and l-Phe during filtration of 100 ppm racemate solution were 0.1287–0.2522 and 0.1174–0.2458 mg m⁻² s⁻¹, respectively. The adsorption selectivity was higher at low concentration. The adsorption selectivities varied from 1.11 to 1.65 and from 1.64 to 2.78 during filtration of 100 and 10 ppm racemate solutions, respectively. In respect to desorption, the fractional difference between d-Phe and l-Phe in the recovered solution from 10 ppm was higher than that from 100 ppm.     

Unusual thionation of a cyclic hexapeptide. Conformational changes and dynamics.

One carbonyl oxygen of the cyclic hexapeptide cyclo(-Gly1-Pro2-Phe3-Val4-Phe5-Phe6-) (A) can be selectively exchanged with sulphur using Yokoyama's reagent. Surprisingly it was not the C=] of Gly1 but that of Phe5 which was substituted and cyclo(-Gly1-Pro2-Phe3-Val4-Phe5 psi [CS-NH]Phe6-) (B) was obtained. Thionation results in a conformational change of the peptide backbone although the C=O of Phe5 and the corresponding C=S are not involved in internal hydrogen bonds. Two isomers in slow exchange, containing a cis Gly1-Pro2 bond in a beta VIa-turn (minor) and a trans Gly-Pro bond in a beta II'-turn (major), were analyzed by restrained molecular dynamics in vacuo and in DMSO as well as using time dependent distance constraints. It is impossible to fit all experimental data to a static structure of each isomer. Interpreting the conflicting NOEs, local segment flexibility is found. MD simulations lead to a dynamic model for each structure with evidence of an equilibrium between a beta I- and beta II-turn about the Val4-Phe5 amide bond in both the cis and trans isomers. Additionally proton relaxation rates in the rotating frame (R1 rho) were measured to verify the assumption of this fast beta I/beta II equilibrium within each isomer. Significant contributions to R1 rho-rates from intramolecular motions were found for both isomers. Therefore it is possible to distinguish between at least four conformers interconverting on different time scales based on NMR data and MD refinement. This work shows that thionation is a useful modification of peptides for conformation-activity investigations.     

Nonpeptide antagonists for kinin receptors

Kinins are a family of small peptides acting as mediators of inflammation and pain in the peripheral and central nervous system. The two main 'kinins' in mammals are the nonapeptide bradykinin (BK, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) and the decapeptide kallidin (KD, [Lys0]-BK, Lys1-Arg2-Pro3-Pro4-Gly5-Phe6-Ser7-Pro8-Phe9- Arg10). Their biological actions are mediated by two distinct receptors, termed B1 and B2. Kinin B and B2 receptor antagonists may be useful drugs endowed with analgesic and anti-inflammatory properties, with potential use in asthma, allergic rhinitis and other diseases. The first nonpeptide kinin B2 receptor antagonist, WIN 64338, was reported in 1993. Despite its low selectivity, the compound provided a reference for pharmacological and modeling studies. Several quinoline and imidazo[1,2-a]pyridine derivatives have been shown by Fujisawa to possess high affinity and selectivity for kinin B2 receptors. Among them, FR 173657 displayed excellent in vitro and in vivo antagonistic activity, while FR 190997 emerged as the first nonpeptide agonist for B2 receptor. Two structurally related Fournier compounds were recently published. Other kinin B2 receptor ligands were obtained by rational design, through library screening or from natural sources. The only example of a nonpeptide kinin B1 receptor ligand has been reported in a patent by Sanofi.     

Tetrazole analogues of cyclolinopeptide A: synthesis, conformation, and biology

Linear and cyclic analogues of cyclolinopeptide A (CLA) with two dipeptide segments (Val(5)-Pro(6) and Pro(6)-Pro(7)) replaced by their tetrazole derivatives were synthesized by the SPPS technique and cyclized using TBTU (O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate) reagent. The conformational properties of the c(Leu(1)-Ile(2)-Ile(3)-Leu(4)-Val(5)-Pro(6)-psi[CN(4)]-Ala(7)-Phe(8)-Phe(9)) were investigated by NMR and computational techniques. The overall solution structure of this cyclic peptide resembles that observed for the CLA in the solid state. These studies of cyclic tetrazole CLA analogue confirm that the 1,5-disubstituted tetrazole ring functions as an effective, well-tolerated cis-amide bond mimic in solution. The peptides were examined for their immunosuppressive activity in the humoral response test. For cyclic analogues the immunosuppressive activity, at low doses, is equal in magnitude to the activity presented by cyclosporin A and native CLA. The conformational and biological data seem indicate that the Pro-Pro-Phe-Phe moiety and the preservation of the CLA backbone conformation are important for immunosuppressive activity.     

New cyclic peptides with osteoblastic proliferative activity from Dianthus superbus

Two new cyclic peptides, dianthins G-H (1 and 2), together with the known dianthin E (3), were isolated from the traditional Chinese medicinal plant Dianthus superbus. The sequences of cyclic peptides 1 and 2 were elucidated as cyclo (-Gly(1)-Pro(2)-Leu(3)-Thr(4)-Leu(5)-Phe(6)-) and cyclo (-Gly(1)-Pro(2)-Val(3)-Thr(4)-Ile(5)-Phe(6)-), on the basis of ESI tandem mass fragmentation analysis, extensive 2D NMR methods and X-ray diffraction. The isolated three compounds all increase proliferation of MC3T3-E1 cells in vitro using MTT method.     

Conformational studies of two bradykinin antagonists by using two-dimensional NMR techniques and molecular dynamics simulations

The solution conformations of two potent antagonists of bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9), [Aca(-1),DArg0,Hyp3,Thi5,DPhe7,(N-Bzl)Gly8]BK (1) and [Aaa(-1),DArg0,Hyp3,Thi5,(2-DNal)7,Thi8]BK (2), were studied by using 2D NMR spectroscopy in DMSO-d6 and molecular dynamics simulations. The NMR spectra of peptide 1 reveals the existence of at least two isomers arising from isomerization across the DPhe7-(N-Bzl)Gly8 peptide bond. The more populated isomer possesses the cis peptide bond at this position. The ratio of cis/trans isomers amounted to 7:3. With both antagonists, the NMR data indicate a beta-turn structure for the Hyp3-Gly4 residues. In addition, for peptide 2, position 2,3 is likely to be occupied by turn-like structures. The cis peptide bond between DPhe7 and (N-Bzl)Gly8 in analogue 1 suggests type VI beta-turn at position 7,8. The molecular dynamics runs were performed on both peptides in DMSO solution. The results indicate that the structure of peptide 1 is characterized by type VIb beta-turn comprising residues Ser6-Arg9 and the betaI or betaII-turn involving the Pro2-Thi5 fragment, whereas peptide 2 shows the tendency towards the formation of type I beta-turn at position 2,3. The structures of both antagonists are stabilized by a salt bridge between the guanidine moiety of Arg1 and the carboxyl group of Arg9. Moreover, the side chain of DArg0 is apart of the rest of molecule and is not involved in structural elements except for a few calculated structures.     

Conformational analysis of a potent SSTR3-selective somatostatin analogue by NMR in water solution.

The three-dimensional structure of a potent SSTR3-selective analogue of somatostatin, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-Agl(8)(N(beta) Me, 2-naphthoyl)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-Agl(8)(N(beta) Me, 2-naphthoyl)]-SRIF) (peptide 1) has been determined by (1)H NMR in water and molecular dynamics (MD) simulations. The peptide exists in two conformational isomers differing mainly by the cis/trans isomerization of the side chain in residue 8. The structure of 1 is compared with the consensus structural motifs of other somatostatin analogues that bind predominantly to SSTR1, SSTR2/SSTR5 and SSTR4 receptors, and to the 3D structure of a non-selective SRIF analogue, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-2Nal(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-2Nal(8)]-SRIF) (peptide 2). The structural determinant factors that could explain selectivity of peptide 1 for SSTR3 receptors are discussed.     

Identification and characterization of the calmodulin-binding domain of neuromodulin, a neurospecific calmodulin-binding protein

Neuromodulin (formerly designated P-57) is an abundant, neural specific, calmodulin-binding protein which exhibits higher affinity for calmodulin in the absence of free Ca2+ than in the presence of free Ca2+. In this study a series of proteolytic fragments of neuromodulin were systematically screened for calmodulin-Sepharose binding activity. A 9-amino acid fragment, designated M1-C1 and having the sequence RGHITRKKL, was identified as the putative CaM-binding domain of neuromodulin. Two heptadecapeptides, designated FP57-Phe and FP57-Trp, were synthesized, each containing the M1-C1 sequence and the four flanking amino acids from each site. The FP57-Trp peptide contained a tryptophan residue in place of the native phenylalanine. Anti-FP57-Phe antibody binding to neuromodulin was inhibited by preincubation of antibodies with excess FP57-Phe. 125I-CaM gel overlay of neuromodulin was inhibited by anti-FP57-Phe antibodies. Addition of CaM to FP57-Trp increased peptide tryptophanyl fluorescence. In the presence of Ca2+, the stoichiometry of the FP57-Trp.CaM complex was 1:1, FP57-Trp binding to CaM was competitive with neuromodulin. The Ca2+-independent dissociation constant of the FP57-Phe.CaM complex was 0.41 microM. The Ca2+-dependent affinity of the complex could not be measured directly but appeared to be significantly greater than the Ca2+-independent affinity.     

Mechanism of action of aspartyl proteinases. VI. Nonvalent enzyme-inhibitory and enzyme-substrate complexes of the aspartyl proteinase rhizopus pepsin

The structure of a complex of rhizopuspepsin, a fungal aspartyl protease, with Pro1-Phe2-His3-Phe4-psi[CH2-NH]-Phe5-Val6, its substrate-like inhibitor, was calculated by theoretical conformational analysis. The search for energetically favorable conformational variants of the ligand structure was based on the fragmental approach using the dynamic library of peptide fragments, which were successively extended in the potential field of the protein. The root-mean-square deviation of atom positions in the calculated and experimental inhibitor conformations was 0.56 A. A similar approach was used to model a noncovalent complex of rhizopuspepsin with Pro1-Phe2-His3-Lys4-Phe5-Val6, its specific substrate. As a result, two isoenergetic structures of the complex with different arrangements of the cleavable peptide group and a nucleophilic water molecule were calculated. The possibility of the achieving each of these conformations during the catalytic act is considered. It is shown that there are no structural prerequisites for the distortion of the cleavable bond in the active site of the enzyme. On the basis of the resulting structural data, the assumption was made that Asp35 may be protonated at a late stage of formation of the tetrahedral intermediate rather than at the basic state of the complex.     

Topology of proteolipid protein in the myelin membrane of central nervous system. A study using antipeptide antibodies

Peptides according to amino-acid sequences of the N- and C-terminus of lipophilin (proteolipid protein, PLP) (Gly1-Phe15 = 1; Thr261-Phe276 = 6) and of the other four hydrophilic domains (Glu37-Leu60 = 2; Arg97-Leu112 = 3; Gly119-Gly127 = 3A; Trp144-Tyr156 = 3B; Lys191-Ala203 = 4; Asn222-Phe232 = 5) have been synthesized by the solid-phase Fmoc method, linked covalently to keyhole limpet hemocyanin (KLH) and used as antigens. Monospecific antibodies against these antigens were isolated by affinity chromatography. Each antibody recognized its epitope in isolated partially delipidated PLP with the ELISA technique, western blot, thin sections of paraffin embedded rat brains and in the plasma membrane of appropriately fixed/permeabilized rat oligodendrocytes in culture. After fixation with formaldehyde antipeptide 3A antibody stained intact non-permeabilized cells. Therefore the epitope 3A must be located on the extracellular surface of the membrane. This is in full support of our previous biochemical results on the orientation of lipophilin in the myelin membrane.     

Degradation of bradykinin and its metabolites by rat brain synaptic membranes

Bradykinin (BK) (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) was degraded by rat brain synaptic membranes at a rate comparable to that found for Met-enkephalin, but approximately 40 times the rate for vasopressin and oxytocin. The catabolic pathway for BK and its metabolites was elucidated through the use of high performance liquid chromatography for metabolite identification and peptidase inhibitors for blocking specific cleavage sites. BK was hydrolyzed at three sites: at the -Phe5-Ser6- bond by metalloendopeptidase 24.15, at the -Pro7-Phe8- bond by an apparently novel peptidyl dipeptidase, and at the -Phe8-Arg9 bond by a carboxypeptidase B-like enzyme. Each enzyme contributed about equally to BK degradation under the assay conditions used. Some of the resulting metabolites were further hydrolyzed: BK(1-8) to BK(1-7) + Phe by a DFP inhibitable prolyl carboxypeptidase-like enzyme, BK(1-8) to BK(1-5) + BK(6-8) by metalloendopeptidase 24.15, BK(1-7) slowly to BK(1-5) by a second peptidyl dipeptidase which was captopril inhibited, and Phe-Arg to Phe + Arg by a bestatin-inhibited dipeptidase. A number of properties of the individual enzymes were determined including sensitivity to a variety of peptidase inhibitors. These results provide a starting point for investigating the potential physiological role of each enzyme in BK function in the brain.