NMR and simulated annealing investigations of bradykinin in presence of polyphenols

Epidemiological studies have shown that the incidence of some cardiovascular degenerative diseases appears to be lower in populations with regular but moderate drinking of red wine rich in polyphenols. One of the most important properties of polyphenols is to form complexes with proteins. The linear nonapeptide hormone bradykinin (H-Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9-OH) is involved in a variety of physiological processes such as the cardiovascular processes. Thus, the goal of this work was to study the effects of tannins on the peptide structure by NMR investigations and molecular modeling. The results of these investigations show that in the presence of catechin, the peptide conformation is not affected and is in a random coil structure. On the contrary, the peptide structure is modified by the addition of dimeric proanthocyanidin B3 (catechin 4alpha-->8 catechin). The dimer leads to the formation of a large flexible turn between the 6-9 residues. Thus, the biological activities of bradykinin in the presence of polyphenols could be affected.     

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.     

Structural characterization of lipopeptide agonists for the bradykinin B2 receptor

The conformational features of Pam-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PKD) and Pam-Gly(-1)-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PGKD), the Pam-Lys and Pam-Gly-Lys analogues of bradykinin, have been determined by high-resolution NMR in a zwitterionic lipoid environment. Radical-induced relaxation of the (1)H NMR signals was used to probe the topological orientation of the peptides with respect to the zwitterionic lipid interface. The radical-induced relaxation and molecular dynamics (MD) data indicated that the palmitic acid and N-terminal amino acid residues embed into the micelles, while the rest of the polypeptide chain is closely associated with the water-micelle interface. Throughout the entire nuclear Overhauser effect restrained MD simulation, a nonideal type I beta-turn was observed in the C-terminus of PKD between residues 6 and 9, and a gamma-turn was observed in the C-terminus of PGKD between residues 6 and 7. Therefore, the additional glycine has a dramatic effect on the structural preferences of the biologically important C-terminus, an effect brought about by the interaction with the lipid environment. These structural features are correlated to the biological activity at the bradykinin B2 receptor.     

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.     

Isolation of brain endopeptidases: influence of size and sequence of substrates structurally related to bradykinin.

Two thiol-activated endopeptidases with pH optima near pH 7.5 were isolated from the supernatant fraction of rabbit brain homogenates by DEAE-cellulose chromatography, gel filtration and isoelectrofocusing. Peptide bond hydrolysis was measured quantitatively by ion-exchange chromatography with an amino acid analyzer. Brain kininase A hydrolyzes the Phe5-Ser6 peptide bond in bradykinin (Bk), Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9. It is isoelectric near pH 5.2 and has a molecular weight of approximately 71 000. The enzyme also hydrolyzes the Phe-Ser peptide bond in Lys-Bk, Met-Lys-Bk, des-Arg1-Bk, Lys9-Bk, Pro-Gly-Phe-Ser-Pro-Phe-Arg, and Gly-Pro-Phe-Ser-Pro-Phe-Arg, but does not hydrolyze (0.1%) this bond in des-Phe8-Arg9-Bk. Brain kininase B hydrolyzes the Pro7-Phe8 peptide bond in Bk. It is isoelectric at pH 4.9 and has a molecular weight of approximately 68 000. Brain kininase B also hydrolyzes the Pro-Phe bond in Lys-Bk, Met-Lys-Bk, Lys9-Bk, Ser-Pro-Phe-Arg, and Phe-Ser-Pro-Arg. Pretreatment of denatured kininogen with brain kininase A or B did not reduce the amount of trypsin-releasable Bk from this precursor protein, indicating that the Bk sequence, when part of a large protein, is not a substrate for either enzyme. However, kininase A and B hydrolyze the octadecapeptide Gly-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gin-Val. The data show that a large part of the C-terminal portion of bradykinin is important for the brain kininase A activity and, for both enzymes, the size of the peptide and presumably the residues adjacent to the scissle bond are important in determining the rate of peptide bond hydrolysis by these endopeptidases.     

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.     

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.     

Neuropeptide-Metabolizing Peptidases in Neuro-2a Neuroblastoma and C6 Glioma Cells

Mouse Neuro-2a neuroblastoma and rat C6 glioma cloned cells were screened for neuropeptide-metabolizing peptidases using a kininase bioassay combined with a time-course bradykinin-product analysis, and a fluorimetric assay for prolyl endopeptidase. The complementary peptide products Arg1----Phe5/Ser6----Arg9 and Arg1----Pro7/Phe8-Arg9 were released during bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) inactivation by homogenates of Neuro-2a and C6 cells. The 1:1 stoichiometry of the complementary fragments and their high yields, at 10% bradykinin inactivation, demonstrated the sites of hydrolysis. The initial rate of Phe5-Ser6 bond cleavage was six-fold higher than that of the Pro7-Phe8 bond. These sites of cleavage can be attributed to enzymes similar to endopeptidase A (Phe5-Ser6) and prolyl endopeptidase (Pro7-Phe8) on the basis of the specificity and sensitivity to inhibitors of the kininase activity in Neuro-2a and C6 cell homogenates. Kininase and prolyl endopeptidase specific activities (fmol/min/cell) were 10.5 and 12.4 for Neuro-2a, and 1.5 and 2 for C6 homogenate, respectively. The recovery of kininase activity was 2.2-fold higher in the particulate than in the soluble (105,000 g for 1 h) neuronal fraction, whereas the amount of prolyl endopeptidase activity was about the same in both fractions. Kininase and prolyl endopeptidase activities in C6 cells were recovered mostly in the soluble fraction. Prolyl endopeptidase specific activity decreased 10-fold in serum-starved Neuro-2a cultured cells, with no change in activity in similarly treated C6 cells. In contrast, kininase specific activity in both cell types was essentially unaffected on serum-deprivation-induced differentiation.     

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.     

Kininase from the Latrodectus tredecimguttatus venom. Characteristics of the enzyme as a thiol endopeptidase hydrolyzing the -Pro7-Phe8- bond of bradykinin

The nature of the bradykinin (BK)-hydrolyzing (kininase) activity of peptidhydrolase isolated from spider (Latr. tredecimguttatus) venom has been studied. It was found that the BKase activity of the enzyme is fully inhibited by organic mercurials (10(-5)-10(-6) M) as well as by 5,5'-dithiobis(2-nitrobenzoic acid) (10(-7) M); the latter blocks three SH-groups within the enzyme molecule. Serine and metalloproteinase inhibitors have no effect on the kininase activity. Thin-layer chromatography on silicagel revealed that the highly purified enzyme hydrolyzes the -Pro7-Phe8- bond of BK liberating the C-terminal dipeptide, HPhe-ArgOH. Besides, the kininase splits off the C-terminal tripeptide from angiotensin I by hydrolyzing its -Pro7-Phe8-bond. The enzyme does not exhibit any exopeptidase activity with free and N-substituted tri- and pentapeptides. The data obtained suggest that the Latr. tredecimguttatus kininase can be related to thiol endopeptidases hydrolyzing the peptide bonds formed by proline carboxyl.     

Bradykinin analogs containing the 4-amino-2-benzazepin-3-one scaffold at the C-terminus.

High affinity peptide ligands for the bradykinin (BK) B(2) subtype receptor have been shown to adopt a beta-turn conformation of the C-terminal tetrapeptide (H-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH). We investigated the replacement of the Pro(7)-Phe(8) dipeptide moiety in BK or the D-Tic(7)-Oic(8) subunit in HOE140 (H-D-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-D-Tic(7)-Oic(8)-Arg(9)-OH) by 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one templates (Aba). Binding studies to the human B(2) receptor showed a correlation between the affinities of the BK analogs and the propensity of the templates to adopt a beta-turn conformation. The L-spiro-Aba-Gly containing HOE140 analog BK10 has the best affinity, which correlates with the known turn-inducing property of this template. All the compounds did not modify basal inositolphosphate (IP) output in B(2)-expressing CHO cells up to 10 microM concentration. The antagonist properties were confirmed by the guinea pig ileum smooth muscle contractility assay. The new amino-benzazepinone (Aba) substituted BK analogs were found to be surmountable antagonists.     

Ortho-aminobenzoic acid-labeled bradykinins in interaction with lipid vesicles: fluorescence study

The peptide hormone bradykinin (BK) (Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)) and its shorter homolog BK(1-5) (Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)) were labeled with the extrinsic fluorescent probe ortho-aminobenzoic acid (Abz) bound to the N-terminal and amidated in the C-terminal carboxyl group (Abz-BK-NH(2) and Abz-BK(1-5)-NH(2)). The fragment des-Arg(9)-BK was synthesized with the Abz fluorescent probe attached to the 3-amino group of 2,3-amino propionic acid (DAP), which positioned the Abz group at the C-terminal side of BK sequence, constituting the peptide des-Arg(9)-BK-DAP(Abz)-NH(2). The spectral characteristics of the probe were similar in the three peptides, and their fluorescent properties were monitored to study the interaction of the peptides with anionic vesicles of dimyristoylphosphatidylglycerol (DMPG). Time-resolved fluorescence experiments showed that the fluorescence decay of the peptides was best described by double-exponential kinetics, with mean lifetimes values around 8.0 ns in buffer pH 7.4 that increased about 10% in the presence of DMPG vesicles. About a 10-fold increase, compared with the values in aqueous solution, was observed in the steady-state anisotropy in the presence of vesicles. A similar increase was also observed for the rotational correlation times obtained from time-resolved anisotropy decay profiles, and related to the overall tumbling of the peptides. Equilibrium binding constants for the peptide-lipid interaction were examined monitoring anisotropy values in titration experiments and the electrostatic effects were evaluated through Gouy-Chapman potential calculations. Without corrections for electrostatic effects, the labeled fragment Abz-BK(1-5)-NH(2) presented the major affinity for DMPG vesicles. Corrections for the changes in peptide concentration due to electrostatic interactions suggested higher affinity of the BK fragments to the hydrophobic phase of the bilayer.     

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.     

Bradykinin-induced release of prostacyclin and thromboxanes from bovine pulmonary artery endothelial cells. Studies with lower homologs and calcium antagonists

Bovine pulmonary artery endothelial cells, in serum-free culture medium, release small quantities of prostacyclin and thromboxane A2 (3-10 and 0.1-0.3 ng/ml; measured as immunoreactive 6-ketoprostaglandin F1 alpha and thromboxane B2, respectively). The release of these substances is stimulated by up to 20-fold during a 3 min incubation with the vasodilator, bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9). Endothelial cells incubated with [3H]arachidonic acid for 24 h and then exposed to bradykinin for 3 min release 3H into the medium, approximately 65% of which co-chromatographs with 6-ketoprostaglandin F1 alpha and 3% with thromboxane B2. The effects of bradykinin are dose-related and are often discernible when the hormone is used at concentrations believed to occur physiologically (10 pg/ml; approximately 10 pM). Furthermore, the bradykinin molecule must be intact: none of its lower homologs affects the release of prostacyclin, thromboxane A2, or 3H unless used at concentrations (1 microM or higher) unlikely to be achieved in vivo. The release appears to involve calcium uptake and calmodulin: it is abolished by EGTA (5 mM) and inhibited by the 'slow channel' calcium antagonists, verapamil and nifedipine (10-100 microM), and by the calmodulin inhibitor, trifluoperazine (3-30 microM). Our findings suggest that bradykinin exerts some of its hormonal effects by acting on specific receptors possessed by vascular endothelial cells; receptor activation is associated with calcium transport, arachidonate mobilization, and a selective synthesis of prostacyclin, a vasodilator in its own right.     

High-field NMR and circular dichroism solvent-dependent conformational studies of the bradykinin C-terminal tetrapeptide Ser-Pro-Phe-Arg

The conformational properties of the tetrapeptide Ser1-Pro2-Phe3-Arg4, the C-terminal fragment of the nonapeptide hormone bradykinin, have been studied by circular dichroism and two-dimensional NMR techniques. Measurements of coupling constants, NH temperature dependence rates and nuclear Overhauser effects (performed with rotating frame nuclear Overhauser spectroscopy, ROESY) in H2O and CD3OH/D2O (80/20, v/v) reveal different conformations in the corresponding solvent. In aqueous solution the molecule exists in a random conformation or as an average of several conformations in rapid exchange. In CD3OH/D2O, however, the conformation is well-defined. The backbone of the peptide is extended, and the side-chains of Phe3 and Arg4 exhibit unusual rigidity for a peptide of this size. Evidently, the secondary structure is stabilized by a charge interaction between the guanidino group of Arg4 and the terminal carboxyl group, since experiments at various pH's show clearly that the definition of conformation decreases strongly upon protonation of the carboxyl function. A NH3+(Ser1)-COO-(Arg4) salt bridge, as well as any form of turn stabilized by hydrogen bonds can be ruled out with certainty.     

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.     

The importance of the N-terminal beta-turn in bradykinin antagonists

Three peptides, B-10148 (Lys-1-Lys0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6- DF5F7-Oic8; where Hyp is trans-4-hydroxyproline, Igl is alpha-(2-indanyl)glycine, F5F is 2,3,4,5,6-pentafluorophenylalanine and Oic is (3aS,7aS)-octahydroindole-2-carboxylic acid), B-10206 (DArg0-Arg1-Pro2-Hyp3-Gly4-Igl5-Ser6-DF 5F7-Nc7G8-Arg9; where Nc7G is N-cycloheptylglycine) and B- 10284 (Arg1-Pro2-Pro3-Gly4-Phe5-Thr6-DTic7-Oic8- NH2; where Tic is 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), were studied in detail by NMR spectroscopy in 60% CD3OH /40% H2O and modeled by a simulated annealing protocol to determine their solution structure. B-10148, an extremely potent BK B1 receptor antagonist with very high BK B2 receptor antagonist activity, despite lacking a C-terminal Arg, displayed an ideal type II beta-turn from Pro2 to Igl5, as well as a salt bridge between the guanidino group of Arg1 and the carboXylate group of Oic8. B-10206, the most potent B2 antagonist, also displayed an ideal type II beta-turn from Pro2 to Igl5 but secondary structure was not observed at the C-terminal end. The third peptide, B-10284, a des-Arg9 analog with a C-terminal amide and a very potent B2 antagonist, had no definite solution structure. The high activity of these peptides emphasizes the importance of the N-terminal beta-turn and the hydrophobic character at the C-terminus in determining the activity of bradykinin antagonists.     

Structure of a paralytic peptide from an insect, Manduca sexta.

Paralytic peptide 1 (PP1) from a moth, Manduca sexta, is a 23-residue peptide (Glu-Asn-Phe-Ala-Gly-Gly-Cys-Ala-Thr-Gly-Tyr-Leu-Arg-Thr-Ala-Asp-Gly-Arg -Cys-Lys-Pro-Thr-Phe) that was first found to have paralytic activity when injected into M. sexta larvae. Recent studies demonstrated that PP1 also stimulated the spreading and aggregation of a blood cell type called plasmatocytes and inhibited bleeding from wounds. We determined the solution structure of PP1 by two-dimensional 1H NMR spectroscopy to begin to understand structural-functional relationships of this peptide. PP1 has an ordered structure, which is composed of a short antiparallel beta-sheet at residues Tyr11-Thr14 and Arg18-Pro21, three beta turns at residues Phe3-Gly6, Ala8-Tyr11 and Thr14-Gly17, and a half turn at the carboxyl-terminus (residues Lys20-Phe23). The well-defined secondary and tertiary structure was stabilized by hydrogen bonding and side-chain hydrophobic interactions. In comparison with two related insect peptides, whose structures have been solved recently, the amino-terminal region of PP1 is substantially more ordered. The short antiparallel beta-sheet of PP1 has a folding pattern similar to the carboxyl-terminal subdomain of epidermal growth factor (EGF). Therefore, PP1 may interact with EGF receptor-like molecules to trigger its different biological activities.     

Magnetization-transfer n.m.r. investigation of the hydrogen exchange in H2O of the peptide fragment B23-B29 of insulin

Detailed and precise information on the exchanges in water of the peptide hydrogens of the insulin fragment B23-B29 (Gly23-Phe24-Phe25-Tyr26-Thr27-Pro28 -Lys29) has been obtained from magnetization-transfer measurements, and nonlinear least-squares fits of the experimental spectra using the expression for the discrete Fourier transform of a sum of exponentially damped sinusoids. From a comparison of the differential exchange rates with those expected for completely solvent-exposed peptide hydrogens, specific conformational features of the heptapeptide are suggested.