微生物来源的人白细胞弹性蛋白酶抑制剂F01-221A的研究

应用白细胞弹性蛋白酶抑制剂高通量的筛选模型对数千株真菌进行筛选，发现了阳性菌株F01-221。对该菌株的发酵产物进行有机溶剂提取、硅胶柱色谱、Sephadex LH-20柱色谱和ODSHPLC制备等分离纯化，得到单体活性化合物F01-221A，它对人白细胞弹性蛋白酶有较强的抑制活性，其IC50为22．1μmol／L。通过对F01-221A的紫外、质谱、核磁等理化数据分析，确定其与化合物equisetin同质。     

Three‐dimensional solution structure of a disulfide bond isomer of the human insulin‐like growth factor‐I

Abstract: The solution structure of a disulfide bond isomer of human insulin‐like growth factor‐I (IGF‐I) was determined using homonuclear NMR methods. A total of 292 interatomic distance constraints, including 12 related to the disulfide bridges, was used in the distance geometry calculations. The determined structures contain two helical rods corresponding to the sequence regions, Ala⁸–Cys¹⁸ and Leu⁵⁴–Cys⁶¹. Comparison with the previously determined structure of native human IGF‐I revealed partial correspondence of the secondary structure (helices I: Ala⁸–Cys¹⁸ and helices III: Leu⁵⁴–Cys⁶¹) and internal packing. Helix II in native human IGF‐I (residues Gly⁴²–Cys⁴⁸) is disrupted in the isomer. A similar relationship has been described between the structure of native insulin and a homologous disulfide isomer, suggesting that these alternative folds represent general features of insulin‐like sequences. In each case the precision of the distance geometry ensemble is low due in part to resonance broadening and a paucity of NOEs relative to other globular proteins of this size. These observations suggest that tertiary structure of the isomer is not highly ordered. Comparison of the biological activities of native and the disulfide bond isomer of human IGF‐I highlight the importance of Tyr²⁴, Phe²⁵, Phe⁴⁹–Cys⁵² and Phe¹⁶ in binding to the IGF‐I receptor or specific IGFBPs. The relationship of this proposed receptor‐binding surface of human IGF‐I to those of insulin is discussed.     

NMR solution structure of a potent cyclic nonapeptide inhibitor of ICAM‐1‐mediated leukocyte adhesion produced by homologous amino acid substitution

Abstract: We have previously described a disulfide‐linked cyclic nonapeptide (inhibitory peptide‐01, IP01), with the sequence CLLRMRSIC, which binds to intercellular adhesion molecule‐1 (ICAM‐1), and blocks binding to its counter‐structure, the integrin α_Lβ₂ (leukocyte functional antigen‐1, LFA‐1) (Sillerud et al., J. Peptide Res. 62, 2003: 97). We now report the optimization of this peptide by means of single homologous amino acid substitutions to yield a new peptide (IP02‐K6; CLLRMKSAC) which shows an approximately sixfold improvement in inhibitory activity of multivalent leukocyte binding (inhibition constant for 50% inhibition, IC₅₀ = 90 μm ) compared with IP01 (IC₅₀ = 580 μm ). This improvement in activity gives IP02‐K6 potent in vivo activity in a murine model of ischemia reperfusion injury (Merchant et al., Am. J. Physiol. Heart Circ. 284, 2003: H1260). In order to determine the structural features relevant to ICAM‐1‐binding, we have determined the structure of IP02‐K6 using proton nuclear magnetic resonance (NMR) spectroscopy and restrained molecular modeling. In our previously reported study of solution models of IP01, we observed three interconverting conformations during low‐temperature molecular dynamics simulation. In the present study, we find a single conformation of IP02‐K6 similar to one of the previously found conformations of IP01 (family C). In particular, an R4‐S7 β‐turn is present in similar proportions in both conformation C of IP01 and in IP02‐K6; this motif is important in binding to ICAM‐1 because this turn enables the IP02‐K6 backbone to drape over proline‐36 on ICAM‐1. The NMR‐derived solution model of IP02‐K6 was found to dock at the α_Lβ₂‐binding site on ICAM‐1 with no changes in peptide backbone conformation. This docking model displaced five of the 15 α_Lβ₂ residues at the ICAM‐1‐binding site and provided a rationale for understanding the quantitative relationship between IP02‐K6 structure and biologic activity.     

Structure and absolute configuration of pyrophen,a novel pryrone derivative of L-phenylalanine from Aspergillus niger

Pyrophen, a novel 4-methoxy-2-pyrone derivative of L-phenylalanine isolated from cultures of Aspergillus niger, crystallizes in the orthorhombic space group P2₁2₁2₁, with a = 8.918(1), b = 9.199(3), and c = 18.092(4) Å. The structure was solved by direct methods and the absolute configuration assigned by the Bijvoet method. Refinement gave R = 0.045 and S = 1.78 for 1677 reflections with I > 3σ(I). Though numerous other pyrone natural products are known, including other products of A. niger, this is the first report of an amino acid-pyrone derivative.     

新型弹性蛋白酶抑制剂西维来司钠临床前药理及机制研究

西维来司钠是新近研制成功的弹性蛋白酶抑制剂,大量临床前研究资料表明其能选择性地抑制中性粒细胞释放弹性蛋白酶,抑制酶活性,保护肺微血管和上皮细胞,从而保护肺组织,改善呼吸功能,在临床上治疗全身性炎症反应综合征及特发性纤维化等引起的急性肺损伤中显示了良好的应用前景.     

Characterization and NMR solution structure of a novel cyclic pentapeptide inhibitor of pathogenic hantaviruses

Hantavirus-induced diseases such as hantavirus cardiopulmonary syndrome and hemorrhagic fever with renal syndrome are a global health concern. Hantavirus cardiopulmonary syndrome caused by Sin Nombre virus lacks specific therapy and its high mortality makes Sin Nombre virus a potential bioweapon agent. Sin Nombre virus entry into susceptible cells requires expression of alpha(v)beta(3) integrin. We recently reported the sequence of a cyclic nonapeptide that inhibited Sin Nombre virus entry into Vero E6 cells at a level comparable to ReoPro, a Fab fragment of the anti-beta(3) antibody c7E3. Here, we refine the parental peptide, cyclo-[CPFVKTQLC], using alanine scanning and amino acid deletions, by optimizing for viral inhibition. The IC(50) of the resulting peptide, cyclo-[CPFVC], was 267 microM compared with 263 microM for the parental peptide. The solution structure of cyclo-[CPFVC] was determined by two-dimensional nuclear magnetic resonance spectroscopy, revealing the Phe ring in an extended conformation stacked against the Pro ring and containing a beta-turn encompassing Val-4 through Cys-1. As an initial step in identifying interactions between cyclo-[CPFVC] and its target cellular receptor, the refined peptide structure was docked into the ReoPro binding site of integrin beta(3). This structure will provide the basis for designing more potent peptidomimetic therapeutics to prevent Sin Nombre virus entry and treat hantavirus cardiopulmonary syndrome.     

Stereoselective deuterium labeling of proRβ‐protons in the NMR structure determination of a helix‐turn‐helix turn peptide mimic

Abstract: The NMR structure of a small, side‐chain‐cyclized tripeptide mimic of the turn in the helix‐turn‐helix (HTH) motif was determined. The four β‐protons were stereospecifically assigned by stereoselective deuterium replacement of only the proRβ‐protons. All 24 of 30 NOESY cross‐peaks not involving chemically defined or freely rotating protons, and six of seven coupling constants from the P.COSY were used as distance and angle constraints in molecular modeling. MacroModel found 33/1000 structures in the NMR constrained search and 263/1000 structures in the unconstrained search, indicating meaningful constraint by the NMR data. However, the 10 lowest‐energy structures from the unconstrained and constrained searches are very similar, so modeling alone was able to find the experimentally determined structure.     

Lipid‐lowering effects of the squalene epoxidase inhibitor FR194738 in dogs,hamsters, and rats

Squalene epoxidase is a microsomal enzyme that catalyzes the conversion of squalene to 2,3‐oxidosqualene and is an important control site in the cholesterol synthetic pathway. FR194738 is a potent inhibitor of hepatic squalene epoxidase from dogs, hamsters, and rats with IC₅₀ values of 49, 14, and 68 nM, respectively. In dogs, FR194738 at 10 and 32 mg/kg/day decreased serum total cholesterol levels by 26% and 40%, and serum triglyceride levels by 47% and 76%, respectively. Pravastatin, an inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, at 3.2 and 10 mg/kg/day also decreased serum total cholesterol levels by 32% and 36%, and serum triglyceride levels by 56% and 50%, respectively. Both FR194738 and pravastatin preferentially decreased low‐density lipoprotein cholesterol levels among the lipoprotein fractions examined. In hamsters, FR194738 decreased serum total cholesterol levels by 22% and led to a decrease in serum triglyceride levels of 9% at 100 mg/kg/day, whereas pravastatin did not decrease total serum cholesterol levels up to 100 mg/kg/day even though it decreased serum triglyceride levels at doses as low as 3.2 mg/kg/day. In rats, both FR194738 and pravastatin failed to decrease total serum cholesterol levels up to 100 mg/kg/day. FR194738 dose‐dependently decreased serum triglyceride levels by 30%, 41%, and 65% at 10, 32, and 100 mg/kg/day, respectively, whereas pravastatin decreased them only by 19% at 100 mg/kg/day. These results clearly showed a species difference in cholesterol and triglyceride metabolism, and suggest that dogs are the most appropriate animal model for evaluating hypocholesterolemic drugs. FR194738 that induced comparable effects with pravastatin in the dog model may provide another treatment for hypercholesterolemia in men. Drug Dev. Res. 54:202–208, 2001. © 2002 Wiley‐Liss, Inc.     

Conformational studies of a synthetic cyclic decapeptide fragment of rat transforming growth factor-α

The solution conformation of a synthetic cyclic decapeptide [with sequence mimicking the third disulfide loop of rat transforming growth factor-α (rTGF-α)] in deuterated dimethyl sulfoxide was studied by 2D NMR. The determination of solution structures was based on NOE interproton distances, using a combination of distance geometry and simulated annealing protocols. The convergence of the selected structures was evident from the small atomic pairwise root-mean-square deviation values among them. Good agreement was noted between the experimental and simulated NOESY spectra, thereby reflecting the accuracy of the calculated solution structures. Analysis of the structures indicates that the residues Tyr5 and Arg9 exhibit similar side chain orientation as that in the corresponding disulfide loop of human transforming growth factor-α.     

Synthesis,biological activity,and solution structures of a cyclic dodecapeptide from the EGF‐2 domain of blood coagulation factor VII

Abstract: The cyclic dodecapeptide, disulfide‐cyclo‐[H‐Cys‐Val‐Asn‐Glu‐Asn‐Gly‐Gly‐Cys(Acm)‐Glu‐Gln‐Tyr‐Cys‐OH], which corresponds to the 91–102 sequence of the second epidermal growth factor domain of human blood coagulation factor VII, was synthesized using solid‐phase procedures. It was shown to be an inhibitor at the key step in the induction of coagulation by the extrinsic pathway, i.e. the factor VII/tissue factor‐catalyzed activation of coagulation factor X. The solution structure of this peptide was investigated by NMR spectroscopy and was computer‐modeled via molecular mechanics. Structures were calculated based on 112 distance and nine dihedral angle constraints. The resulting backbone structures were classified into two structural subsets: one which exhibited a twisted ‘8’‐shaped folding and another describing an open, circular ‘O’ outline. The local backbone structures of segments Asn³‐Glu⁴‐Asn⁵, Gly⁷‐Cys⁸ and Gln¹⁰‐Tyr¹¹ were well preserved among the two subsets. Apart from the unrestrained N‐ and C‐termini, Gly⁶ and Glu⁹ sites exhibited marked local disorder between the two subsets, suggesting localized flexible hinges likely to govern tertiary structure interconversion between the two subsets. Two transient hydrogen bonds were identified from pH chemical shift titrations by matching the pK_a values of NH and carboxylate groups, which supported the occurrence of the ‘8’ structure, and agreed with temperature coefficients of peptidyl NH resonances. Structure?function relationships of the peptide were discussed in terms of the likely physiological function of the disulfide‐bonded loop in factor VII which the peptide represents.     

Solution structure of a cathelicidin‐derived antimicrobial peptide,CRAMP as determined by NMR spectroscopy

Abstract: CRAMP was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin‐derived antimicrobial peptides. This peptide shows potent antimicrobial activity against gram‐positive and gram‐negative bacteria but no hemolytic activity against human erythrocytes. CRAMP was known to cause rapid permeabilization of the inner membrane of Escherichia coli. In this study, the structure of CRAMP in TFE/H₂O (1 : 1, v/v) solution was determined by CD and NMR spectroscopy. CD spectra showed that CRAMP adopts a mainly α‐helical conformation in TFE/H₂O solution, DPC micelles, SDS micelles and liposomes, whereas it has a random structure in aqueous solution. The tertiary structure of CRAMP in TFE/H₂O (1 : 1, v/v), as determined by NMR spectroscopy, consists of two amphipathic α‐helices from Leu⁴ to Lys¹⁰ and from Gly¹⁶ to Leu³³. These two helices are connected by a flexible region from Gly¹¹ to Gly¹⁶. Previous analysis of series of fragments composed of various portion of CRAMP revealed that an 18‐residue fragment with the sequence from Gly¹⁶ to Leu³³ was found to retain antibacterial activity. Therefore, the amphipathic α‐helical region from Gly¹⁶ to Leu³³ of CRAMP plays important roles in spanning the lipid bilayers as well as its antibiotic activity. Based on this structure, novel antibiotic peptides having strong antibiotic activity, with no hemolytic effect will be developed.     

Conformational comparison of µ‐selective endomorphin‐2 with its C‐terminal free acid in DMSO solution,by 1H NMR spectroscopy and molecular modeling calculation

Abstract: In order to make clear the structural role of the C‐terminal amide group of endomorphin‐2 (EM2, H‐Tyr‐Pro‐Phe‐Phe‐NH₂), an endogenous µ‐receptor ligand, in the biological function, the solution conformations of endomorphin‐2 and its C‐terminal free acid (EM2OH, H‐Tyr‐Pro‐Phe‐Phe‐OH), studied using two‐dimensional ¹H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr‐Pro ω bond in a population ratio of ≈ 1 : 2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton?proton distance constraints derived from the ROE cross‐peaks. These energy‐minimized conformers, which were all in the φ torsion angles estimated from J_NHCαH coupling constants within ± 30°, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro²–Phe³ moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the ‘bow’‐shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C‐ and N‐terminal groups. These results indicate clearly that the substitution of carboxyl group for C‐terminal amide group makes the peptide flexible. The conformational requirement for µ‐receptor activation has been discussed based on the active form proposed for endomorphin‐1 and by comparing conformational features of EM2 and EM2OH.     

Combined 1H‐NMR and Molecular Dynamics Studies on Conformational Behavior of a Model Heptapeptide,GRGDSPC

Among various strategies, the de novo design and in silico approaches are being used to develop the short peptides, models of modified peptides, and mimetics as clinically useful drugs with improved stability and bioavailability. The resulting models will help to isolate the factors behind the folded structure formation and contribute useful information about de novo peptide design. The combined ¹H‐NMR spectroscopic and molecular dynamics methods were used to investigate the conformational behavior of an Arg‐Gly‐Asp (RGD)‐containing peptide, GRGDSPC, the cell‐binding heptapeptide of extracellular matrix protein, fibronectin. The formation of two fused weak β‐turns of type II (HB, 4→1) and type II’ (HB, 7→4) from simulation studies has been consistent with NMR data. The sustainable ‘S’‐shaped molecular structure (which remained unchanged during the entire simulation) and the conformational transitions due to interconversions between multiple turns initiated at Asp⁴, Ser⁵, and Cys⁷ imply that the peptide is flexible in nature. Thus, the model of ‘S’‐shaped structure with flexible multiple turns for GRGDSPC peptide may provide the structural rationale for antagonistic properties of this heptapeptide toward the treatment of integrin‐mediated cellular abnormal behaviors such as thrombosis and metastasis.     

Peptides derived from human C-reaetive protein inhibit the enzymatic activities of human leukocyte elastase and cathepsin G: use of overlapping peptide sequences to identify a unique inhibitor

Ten overlapping 15-mer peptides, spanning the entire inner disulfide loop of human C-reactive protein (residues 36-97), were used to isolate a potent inhibitor of the enzymes human leukocyte elastase and human leukocyte cathepsin G, which are associated with chronic inflammatory tissue damage. In contrast to the inability of intact C-reactive protein to inhibit both enzymes, the synthetic peptide E₆₂ILIFWSKDIGYSFT₇₆ inhibited leukocyte elastase (K_i= 0.18 μm ) and cathepsin G (K_i= 0.25 μm ) at concentrations far lower than the acute-phase concentration of C-reactive protein. Several peptide-enzyme binding motifs were elucidated by structure-function studies, with the Glu₆₂ residue being crucial in establishing long-range subsite interactions. Peptides derived from C-reactive protein, which may be generated in vivo by neutrophil-mediated proteolysis as part of a complex regulatory homeostatic mechanism, may play an important role in regulating the activity of matrix-degrading enzymes, specifically at sites of inflammation. The present results thus may shed additional insight on the physiological functions of the major acute-phase reactant C-reactive protein, and perhaps be used as a basis for the design of novel therapeutic substances.     

Relationship between temporary inhibition and structure of disulfide‐linkage analogs of marinostatin,a natural ester‐linked protein protease inhibitor

Abstract: A 12‐residue marinostatin [MST(1–12): ¹FATMRYPSDSDE¹²] which contains two ester linkages of Thr³–Asp⁹ and Ser⁸–Asp¹¹ strongly inhibits subtilisin. In order to study the relationship between the inhibitory activity, structure, and stability of MST, MST analogs were prepared by changing ester linkages to a disulfide linkages. The analogs without the disulfide linkage between 3 and 9 positions lost their inhibitory activity. The K_i value of 1SS(C³–C⁹) (¹FACMRYPSCSDE¹²), which has a single disulfide linkage of Cys³–Cys⁹ was comparable with those of MST(1–12) and MST‐2SS (¹FACMRYPCCSCE¹²), a doubly linked analog of Cys³–Cys⁹ and Cys⁸–Cys¹¹. However, 1SS(C³–C⁹) and MST‐2SS showed temporary inhibition, but not MST(1–12): These analogs were inactivated after incubation with subtilisin for 30 min, and were specifically hydrolyzed at the reactive site. ¹H NMR study showed that 1SS(C³–C⁹) has two conformations, which contain a cis‐ (70%) or trans‐ (30%) Pro residue, while MST‐2SS as well as MST(1–12) takes a single conformation containing only a cis‐Pro residue. Hydrogen–deuterium exchange rate of the Arg⁵ (P1′) NH proton of the MST analogs was about 100 times faster than that of MST(1–12). These results indicate that the linkage between the positions 8 and 11 plays a role for fixing the cis‐conformation of the Pro⁷ residue, and that the linkage between 3 and 9 is indispensable for the inhibition, but not enough for stable protease‐inhibitor complex.     

Solution structure of the B-chain of insulin as determined by 1H NMR spectroscopy Comparison with the crystal structure of the insulin hexamer and with the solution structure of the insulin monomer

The solution structure of the isolated B-chain of bovine insulin has been determined by ¹H NMR spectroscopy combined with simulated annealing calculations. Complete sequence-specific assignments for the proton resonances are reported together with a set of 309 NOES used in the structure calculations. Chemical-shift variations from random coil values provide support for the existence of helical regions in the polypeptide chain, as do a characteristic series of d_αp(i,i+ 3) NOES from residues B8 to B17. While there is some evidence for a limited degree of conformational averaging over the helical region, in general the helix is relatively well defined and corresponds closely to the helical region seen in the X-ray crystal structure of the insulin hexamer. Other similarities with the crystal structure include turn-like conformations at the carboxy terminal end of the helix and extended strands at both the amino and carboxy termini of the peptide. These similarities between the crystal structure and the isolated B-chain suggest that this peptide has intrinsic folding properties, which allow it to adopt its characteristic structure in intact insulin without the need for extensive cooperative interactions with the A-chain. Despite these general similarities, an important difference between the isolated B-chain and the intact protein occurs in the carboxy terminal region. This region appears significantly more mobile in the isolated B-chain. As a conformational change involving the carboxy terminus has been implicated in receptor binding, the current study of the isolated B-chain provides valuable information on the extent of this region's intrinsic mobility. © Munksgaard 1995.     

The cytoplasmic helix of cannabinoid receptor CB2, a conformational study by circular dichroism and 1H NMR spectroscopy in aqueous and membrane‐like environments

Abstract: The cytoplasmic helix domain (fourth cytoplasmic loop, helix 8) of numerous G protein‐coupled receptors (GPCRs) such as rhodopsin and the β‐adrenergic receptor exhibit unique structural and functional characteristics. Computer models also predict this structure for the cannabinoid CB2 receptor, another member of the GPCR superfamily. In our study, a peptide corresponding to helix 8 of the CB2 receptor was synthesized chemically and its secondary structure determined by circular dichroism (CD) and ¹H NMR spectroscopy. NMR and CD revealed an α‐helical structure in this region in both dodecylphosphocholine micelles and dimethylsulfoxide, in contrast to a random coil configuration found in aqueous solvent. This finding is in good agreement with other previous GPCR structural studies including X‐ray crystallography. By combining our finding with other studies, we further hypothesize that the amphipathic nature of helix 8 can play a significant role in the function and regulation of CB receptors as well as other GPCRs in general.     

Structure elucidation of the designer drug N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide and the relevance of predicted 13C NMR shifts – a case study

The detailed structure elucidation process of the new cannabimimetic designer drug, N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide, with a highly substituted pyrazole skeleton, using nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric (MS) techniques is described. After a first analysis of the NMR spectra and comparison with 48 possible pyrazole and imidazole structures, a subset of six positional isomeric pyrazoles and six imidazoles remained conceivable. Four substituents of the heterocyclic skeleton were identified: a proton bound to a pyrazole ring carbon atom; a 5‐fluoropentyl group; a 4‐fluorophenyl substituent; and a carbamoyl group, which is N‐substituted with a methyl residue carrying a tert.‐butyl and a carbamoyl substituent. The 5‐fluoropentyl residue is situated at the nitrogen ring atom. Additional NMR experiments like the ¹H,¹³C HMBC were performed, but due to the small number of signals based on long‐range couplings, the comparison of predicted and observed ¹³C chemical shifts became necessary. The open access Internet shift prediction programs NMRDB, NMRSHIFTDB2, and CSEARCH were employed for the prediction of ¹³C shift values which allowed an efficient and unambiguous structure determination. For the identified N‐(1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl)‐1‐(5‐fluoropentyl)‐3‐(4‐fluorophenyl)‐pyrazole‐5‐carboxamide, the best agreement between predicted ¹³C shifts and the observed chemical shifts and long‐range couplings for the pyrazole ring carbon atoms, with a standard error of about 2 ppm, was found with each of the predictions. For the comparison of measured and predicted chemical shifts model compounds with simple substituents proved helpful. The identified compound is a homologue of AZ‐037 which is offered by Internet suppliers. Copyright © 2015 John Wiley & Sons, Ltd.     

Solution structure of a novel ETB receptor selective agonist ET1–21[Cys(Acm)1,15, Aib3,11, Leu7] by nuclear magnetic resonance spectroscopy and molecular modelling

The solution structure of a biologically active modified linear endothelin-1 analogue, ET1–21[Cys(Acm)^1,15, Aib^3,11, Leu⁷], has been determined for the first time by two-dimensional nuclear magnetic resonance spectroscopy in a methanol-d₃/water solvent mixture. Out of approximately one hundred linear peptide analogues tested by biological assay, this peptide, together with a dozen others, showed significant ET_B selective agonist activity. Here we report the solution structure of an ET_B selective agonist of a full-length, synthetic linear endothelin analogue. The calculated structures indicate that the peptide adopts an α-helical conformation between residues Ser⁵–His¹⁶, whilst both N- and C-termini show no preferred conformation. These results suggest that the disulphide bridges normally associated with endothelin and sarafotoxin peptides may not necessarily be important for either ET_B receptor binding activity or the formation of a helical conformation in solution.     

NMR solution structure of the receptor binding domain of Pseudomonas aeruginosa pilin strain P1. Identification of a β-turn

The solution structure of the peptide antigen from the receptor binding domain of Pseudomonas aeruginosa strain P1 has been determined using two-dimensional ¹H NMR techniques. Ensembles of solution conformations for the trans form of this 23-residue disulfide bridged peptide have been generated using a simulated annealing procedure in conjunction with distance and torsion angle restraints derived from NMR data. Comparison of the NMR-derived solution structures of the P1 peptide with those previously determined for the 17-residue PAK, PAO and KB7 strain peptides [Mclnnes, C., et al. (1993) Biochemistry 32 , 13432–13440; Campbell, A.P., et al. (1995) Biochemistry 34 , 16255–16268] reveals the common structural motif of a β-turn, which may be the necessary structural requirement for recognition of a common cell surface receptor and a common cross-reactive antibody to which all four strains bind. The importance of this conserved β-turn in the PAK, PAO, KB7 and P1 peptides is discussed with regard to the design of a synthetic peptide vaccine effective against multiple strains of Pseudomonas aeruginosa infections.