The pseudo-beta I-turn. A new structural motif with a cis peptide bond in cyclic hexapeptides

Synthesis and conformational analysis of three cyclic hexapeptides cyclo(-Gly1-Pro2-Phe3-Val4-Xaa5-Phe6), Xaa = Phe (I), D-Phe (II) and D-Pro (III), were carried out to examine the influence of proline on the formation of reverse turns and the dynamics of hydrophobic peptide regions. Assignment of all 1H and 13C resonances was achieved by homo- and heteronuclear 2D-NMR techniques (TOCSY, ROESY, HMQC, HMQC-TOCSY and HMBCS-270). The conformational analysis is based on interproton distances derived from ROESY spectra and homo- and heteronuclear coupling constants (E.COSY, HETLOC and HMBCS-270). For structural refinements restrained molecular dynamics (MD) simulations in vacuo and in DMSO were performed. Each peptide exhibits two conformations in DMSO solution due to cis-trans isomerism about the Gly-Pro peptide bond. Surprisingly the cis-Gly-Pro segment in the minor isomers is not involved in a beta VI-turn, but forms a turn structure with cis-Gly-Pro in the i and i + 1 positions. Although no stabilizing hydrogen bond is found in this turn, the phi- and psi-angles closely correspond to a beta I-turn [Pro2: phi(i + 1) -60 degrees, psi(i + 1) -30 degrees; Phe3: phi(i + 2) -100 degrees, psi(i + 2) -50 degrees]. Hence we call this structural element a pseudo-beta I-turn. As expected, in the dominating all-trans isomers proline occupies the i + 1 position of a standard beta I-turn. Therefore, cis-trans isomerization of the Gly1-Pro2 amide bond only induces a local conformational rearrangement, with minor structural changes in other parts of the molecule. However, the geometry of the other regions is affected by the chirality of the i + 1 amino acid for both isomers (beta I for Phe5, beta II' for D-Phe5 or D-Pro5).     

Helix-forming tendencies of amino acids depend on their sequence contexts: tripeptides AFG and FAG show incipient beta-bulge formation in their crystal structures

Many of the theoretical methods used for predicting the occurrence of alpha-helices in peptides are based on the helical preferences of amino acid monomer residues. In order to check whether the helix-forming tendencies are based on helical preferences of monomers only or also on their sequence contexts, we synthesized permuted sequences of the tripeptides GAF, GAV, and GAL that formed crystalline helices with near alpha-helical conformation. The tripeptides AFG and FAG formed good crystals. The x-ray crystallographic studies of AFG and FAG showed that though they contain the same amino acids as GAF but in different sequences, they do not assume a helical conformation in the solid state. On the other hand, AFG and FAG, which contain the same amino acids but in a different sequence, exhibit nearly the same backbone torsion angles corresponding to an incipient formation of a beta-bulge, and exhibit nearly identical unit cells and crystal structures. Based on these results, it appears that the helix-forming tendencies of amino acids depend on the sequence context in which it occurs in a polypeptide. The synthetic peptides AFG (L-Ala-L-Phe-Gly) and FAG (L-Phe-L-Ala-Gly), C14H19N3O4, crystallize in the orthorhombic space group P2(1)2(1)2(1), with a = 5.232(1), b = 14.622(2), c = 19.157(3) A, Dx = 1.329 g cm-3, Z = 4, R = 0.041 for 549 reflections for AFG, and with a = 5.488(2), b = 14.189(1), c = 18.562(1) A, Dx = 1.348 g cm-3, Z = 4, R = 0.038 for 919 reflections for FAG. Unlike the other tripeptides GAF, GGV, GAL, and GAI, the crystals of AFG and FAG do not contain water molecule, and the molecules of AFG aor FAG do not show the helical conformation. The torsion angles at the backbone of the peptide are psi 1 = 144.5(5) degrees; phi 2, psi 2 = -98.1(6) degrees, -65.2(6) degrees; phi 3, psi 13, psi 31 = 154.1(6) degrees, -173.6(6) degrees, 6.9(8) degrees for AFG; and psi 1 = 162.6(3) degrees; phi 2, psi 2 = -96.7(4) degrees, -46.3(4) degrees; phi 3, psi 13, psi 31 = 150.1(3) degrees, -168.7(3) degrees, 12.2(5) degrees for FAG. The conformation angles (phi, psi) for residues 2 and 3 for both AFG and FAG show incipient formation of an beta-bulge.     

Non-proline cis peptide bonds in proteins

In a non-redundant set of 571 proteins from the Brookhaven Protein Data Base, a total of 43 non-proline cis peptide bonds were identified. Average geometrical parameters of the well-defined cis peptide bonds in proteins determined at high resolution show that some parameters, most notably the bond angle at the amide bond nitrogen, deviate significantly from the corresponding one in the trans conformation. Since the same feature was observed in cis amide bonds in small molecule structures found in the Cambridge Structural Data Base, a new set of parameters for the refinement of protein structures containing non-Pro cis peptide bonds is proposed.A striking preference was observed for main-chain dihedral angles of the residues involved in cis peptide bonds. All residues N-terminal and most residues C-terminal to a non-Pro cis peptide bond (except Gly) are located in the beta-region of a phi/psi plot. Also, all of the few C-terminal residues (except Gly) located in the alpha-region of the phi/psi plot constitute the start of an alpha-helix in the respective structure. In the majority of cases, an intimate side-chain/side-chain interaction was observed between the flanking residues, often involving aromatic side-chains. Interestingly, most of the cases found occur in functionally important regions such as close to the active site of proteins. It is intriguing that many of the proteins containing non-proline cis peptide bonds are carbohydrate-binding or processing proteins. The occurrence of these unusual peptide bonds is significantly more frequent in structures determined at high resolution than in structures determined at medium and low resolution, suggesting that these bonds may be more abundant than previously thought. On the basis of our experience with the structure determination of coagulation factor XIII, we developed an algorithm for the identification of possibly overlooked cis peptide bonds that exploits the deviations of geometrical parameters from ideality. A few likely candidates based on our algorithm have been identified and are discussed.     

Coexistence of folded and extended conformations of a tripeptide containing alpha, alpha -di-n-propylglycine in crystals

The crystal structure of the tripeptide Boc-Leu-Dpg-Val-OMe (Dpg, alpha, alpha -di-n-propylglycine) reveals the coexistence of two distinct backbone conformations. In molecule A the Dpg residue adopts a fully extended conformation (phi = 76.0 degrees, psi = 180.0 degrees) while in molecule B a left handed helical conformation (phi = 62.8 degrees, psi = 39.6 degrees) is observed. Molecule B adopts a folded structure corresponding to a highly distorted Type II beta-turn conformation, which lacks an intramolecular 4 -> 1 hydrogen bond. In contrast, molecule A has an open, extended conformation. The results demonstrate that both fully extended and helical conformations are energetically accessible to the Dpg residue.     

Role of Epstein-Barr virus in fine-needle aspirates of metastatic neck nodes in the diagnosis of nasopharyngeal carcinoma

The crystal structure of the peptide Boc-Phe-Val-OMe determined by X-ray diffraction methods is reported in this paper. The crystals grown from aqueous methanol are orthorhombic, space group P2(1)2(1)2(1),a = 11.843(2), b = 21.493(4), c = 26.676(4) A3 and V = 6790 A3. Data were collected on a CAD4 diffractometer using MoK alpha radiation (lambda = 0.7107 A) up to Bragg angle theta = 26 degrees. The structure was solved by direct methods and refined by a least-squares procedure to an R value of 6.8% for 3288 observed reflections. There are three crystal-lographically independent peptide molecules in the asymmetric unit. All the three molecules exhibit extended conformation. The sidechain of the Val2 residue shows two different conformations. The conformation of the peptide Boc-Phe-Val-OMe is compared with the conformation of Ac-delta Phe-Val-OH. It is observed that while Boc-Phe-Val-OMe exhibits an extended conformation, Ac-delta Phe-Val-OH shows a folded conformation. The results of this comparison highlight the conformation constraining property of the delta Phe residue. Interestingly, even though Boc-Phe-Val-OMe and Ac-delta Phe-Val-OH are conformationally different, they exhibit similar packing patterns in the solid state.     

Orientation of the saccharide chains of glycolipids at the membrane surface: conformational analysis of the glucose-ceramide and the glucose-glyceride linkages using molecular mechanics (MM3)

Preferred conformations of the saccharide-ceramide linkage of glucosylceramides with different ceramide structures (normal and hydroxy fatty acids) were investigated by molecular mechanics (MM3) calculations and compared with conformational features obtained for glucosylglycerolipids (diacyl and dialkyl analogues). Relaxed energy map calculations with MM3 were performed for the three bonds (C1'-O1-C1-C2, torsion angles phi, psi, and theta 1) of the glucose-ceramide/diglyceride linkage at different values of the dielectric constant. For the phi torsion of the glycosidic C1'-O1 bond the calculations show a strict preference for the +sc range whereas the psi/theta 1 energy surface is dependent on the structure of the lipid moiety as well as on the dielectric constant (epsilon). Calculations performed on glucosylceramide with normal and hydroxy fatty acids at epsilon = 4 (bilayer subsurface conditions) show three dominating conformers (psi/theta 1 = ap/-sc, -sc/ap, and ap/ap). The ap/-sc conformer, which represents the global energy minimum, is stabilized by polar interactions involving the amide group. The +sc rotamer of theta 1 is unfavored in sphingolipids due to a Hassel-Ottar effect involving the sphingosine O3 and O1 oxygen atoms. Comparative calculations on glycosylglycerolipid analogues (ester and ether derivatives) show a distinct preference for the ap rotamer of theta 1. An evaluation of the steric hindrance imposed by the surrounding membrane surface shows that in a bilayer arrangement the range of possible conformations for the saccharide-lipid linkage is considerably reduced. The significance of preferred conformations of the saccharide-ceramide linkage for the presentation and recognition of the saccharide chains of glycosphingolipids at the membrane surface is discussed.     

Potential photosensitizers for photodynamic therapy. IV. Photophysical and photochemical properties of azaporphyrin and azachlorin derivatives

The photophysical and photochemical properties of a 5-azaprotoporphyrin derivative ([5]AZPP), a zinc-15-azaporphyrin derivative (Zn-[15]AZIDP) and an E-Z isomeric mixture of a 5-azachlorin derivative ([5]AZCH) were studied in various solvents. The quantum yields of fluorescence phi F0, S1-T1 intersystem crossing phi T0 and singlet oxygen (1 delta g) formation phi delta were measured and the Stern-Volmer constants for the quenching of the S1 states by oxygen and the rate constants of quenching of O2(1 delta g) by the different azaporphyrinoid compounds were obtained. The fluorescence quantum yield (phi F0 = 0.23), the strong absorption in the red (lambda max = 674 nm, epsilon max = 66,000 M-1 cm-1) and the high value of the quantum yield for singlet oxygen (1 delta g) formation (phi delta = 0.65) observed for [5]AZCH recommend azachlorin derivatives as potential markers and photosensitizers for tumour therapy.     

Solution structure of the superactive monomeric des-[Phe(B25)] human insulin mutant: elucidation of the structural basis for the monomerization of des-[Phe(B25)] insulin and the dimerization of native insulin

The three-dimensional solution structure of des-[Phe(B25)] human insulin has been determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Thirty-five structures were calculated by distance geometry from 581 nuclear Overhauser enhancement-derived distance constraints, ten phi torsional angle restraints, the restraints from 16 helical hydrogen bonds, and three disulfide bridges. The distance geometry structures were optimized using simulated annealing and restrained energy minimization. The average root-mean-square (r.m.s.) deviation for the best 20 refined structures is 1.07 angstroms for the backbone and 1.92 angstroms for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are more well defined, with r.m.s. deviations of 0.64 angstroms for the backbone and 1.51 angstroms for all atoms. It is found that the des-[Phe(B25)] insulin is a monomer under the applied conditions (4.6 to 4.7 mM, pH 3.0, 310 K), that the overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer of native insulin are preserved, and that the conformation-averaged NMR solution structure is close to the structure of molecule 1 in the hexamer. The structure reveals that the lost ability of des-[Phe(B25)] insulin to self-associate is caused by a conformational change of the C-terminal region of the B-chain, which results in an intra-molecular hydrophobic interaction between Pro(B28) and the hydrophobic region Leu(B11)-Leu(B15) of the B-chain alpha-helix. This interaction interferes with the inter-molecular hydrophobic interactions responsible for the dimerization of native insulin, depriving the mutant of the ability to dimerize. Further, the structure displays a series of features that may explain the high potency of the mutant on the basis of the current model for the insulin-receptor interaction. These features are: a change in conformation of the C-terminal region of the B-chain, the absence of strong hydrogen bonds between this region and the rest of the molecule, and a relatively easy accessibility to the Val(A3) residue.     

Conformational changes due to vicinal glycosylation: the branched alpha-L-Rhap(1-2)[beta-D-Galp(1-3)]-beta-D-Glc1-OMe trisaccharide compared with its parent disaccharides

Conformations of the alpha-L-Rhap(1-2)-beta-D-Glc1-OMe and beta-D-Galp(1-3)-beta-D-Glc1-OMe disaccharides and the branched title trisaccharide were examined in DMSO-d6 solution by 1H-nmr. The distance mapping procedure was based on rotating frame nuclear Overhauser effect (NOE) constraints involving C- and O-linked protons, and hydrogen-bond constraints manifested by the splitting of the OH nmr signals for partially deuteriated samples. An "isotopomer-selected NOE" method for the unequivocal identification of mutually hydrogen-bonded hydroxyl groups was suggested. The length of hydrogen bonds thus detected is considered the only one motionally nonaveraged nmr-derived constraint. Molecular mechanics and molecular dynamics methods were used to model the conformational properties of the studied oligosaccharides. Complex conformational search, relying on a regular phi, psi-grid based scanning of the conformational space of the selected glycosidic linkage, combined with simultaneous modeling of different allowed orientations of the pendant groups and the third, neighboring sugar residue, has been carried out. Energy minimizations were performed for each member of the phi, psi grid generated set of conformations. Conformational clustering has been done to group the minimized conformations into families with similar values of glycosidic torsion angles. Several stable syn and anti conformations were found for the 1-->2 and 1-->3 bonds in the studied disaccharides. Vicinal glycosylation affected strongly the occupancy of conformational states in both branches of the title trisaccharide. The preferred conformational family of the trisaccharide (with average phi, psi values of 38 degrees, 17 degrees for the 1-->2 and 48 degrees, 1 degree for the 1-->3 bond, respectively) was shown by nmr to be stabilized by intramolecular hydrogen bonding between the nonbonded Rha and Gal residues.     

Motional dynamics of residues in a beta-hairpin peptide measured by 13C-NMR relaxation

Structurally characterizing partially folded peptides is problematic given the nature of their transient conformational states. 13C-NMR relaxation data can provide information on the geometry of bond rotations, motional restrictions, and correlated bond rotations of the backbone and side chains and, therefore, is one approach that is useful to assess the presence of folded structure within a conformational ensemble. A peptide 12mer, R1GITVNG7KTYGR12, has been shown to partially fold in a relatively stable beta-hairpin conformation centered at NG. Here, five residues, G2, V5, G7, Y10, G11, were selectively 13C-enriched, and 13C-NMR relaxation experiments were performed to obtain auto- and cross-correlation motional order parameters, correlation times, bond rotation angular variances, and bond rotational correlation coefficients. Our results indicate that, of the three glycines, G7 within the hairpin beta-turn displays the most correlated phi(t),psi(t) rotations with its axis of rotation bisecting the angle defined by the H-C-H bonds. These positively correlated bond rotations give rise to "twisting" type motions of the HCH group. V5 and Y10 phi,psi bond rotations are also positively correlated, with their CbetaCalphaH groups undergoing similar "twisting" type motions. Motions of near-terminal residues G2 and G11 are less restricted and less correlated and are best described as wobbling-in-a-cone. V5 and Y10 side-chain motions, aside from being highly restricted, were found to be correlated with phi,psi bond rotations. At 303 K, where the hairpin is considered "unfolded," the peptide exists in a transient, collapsed state because backbone and side-chain motions of V5, G7, and Y10 remain relatively restricted, unlike their counterparts in GXG-based tripeptides. These results provide unique information toward understanding conformational variability in the unfolded state of proteins, which is necessary to solve the protein folding problem.     

Stereochemical punctuation marks in protein structures: glycine and proline containing helix stop signals

An analysis on the nature of alpha-helix stop signals has been carried out, using a dataset of 1057 helices identified from 250 high resolution (相似文献   

Multiple solution conformations of the integrin-binding cyclic pentapeptide cyclo(-Ser-D-Leu-Asp-Val-Pro-). Analysis of the (phi, psi) space available to cyclic pentapeptides

The aqueous solution structure of the cyclic pentapeptide cyclo(-Ser-D-Leu-Asp-Val-Pro-) has been determined by two-dimensional 1H-NMR spectroscopy, combined with a conformational search and distance-geometry calculations. As many as five conformers in slow exchange were observed, and the rate of interconversion between components was measured from the build-up rates of exchange peaks. NMR data allowed the structures of the two predominant conformers to be determined. The major component (66%) contained a cis-proline as part of a type-VIa2 beta-turn encompassing residues Asp-Val-cis-Pro-Ser. The second component (16%) contained only trans-amide bonds, and a type-VIII beta-turn formed by residues Val-Pro-Ser-D-Leu. These structures are discussed in relation to the (phi, psi), space available to the cyclic pentapeptide, determined by a conformational search, and in relation to previously published cyclic-pentapeptide structures. The molecule exhibits activity in a scintillation-proximity assay for the inhibition of the interaction between the integrin very-late antigen-4 (VLA-4; alpha 4 beta 1) and vascular-cell-adhesion molecule-1 (VCAM-1). The structure/activity relationship of the LDV sequence is discussed and related to the recently published X-ray structure of VCAM-1. The relevance of the work to the design of anti-inflammatory drugs is discussed.     

Main-chain conformational features at different conformations of the side-chains in proteins

An analysis of the known protein structures has shown that the main-chain torsion angles, phi and psi of a residue can be affected by the side-chain torsion angle, chi1. The (chi1, psi) plot of all residues (except Gly, Ala and Pro) show six distinct regions where points are concentrated-although some of these regions are nearly absent in specific cases. The mean of these clusters can show a shift along the psi axis by as much as 30 degrees as chi1 is changed from around 180 to -60 to 60 degrees. Because of the lesser steric constraint points are more diffused along the psi axis when chi1 is approximately -60 degrees. Although points are more spread out along the phi axis in the (chi1, phi) plot, the dependence of phi on chi1 shows up in a shortened phi range (by about 30 degrees) when chi1 is around -60 degrees, and a distinct tendency of clustering of points into two regions when chi1 is approximately equal to 60 degrees, especially for the aromatic residues. Based on the dependence of the backbone conformation on its side-chain the 17 amino acids can be grouped into five classes: (i) aliphatic residues branched at the Cbeta position (although Thr is atypical), (ii) Leu (branched at the Cgamma position), (iii) aromatic residues (Trp can show some deviations), (iv) short polar residues (Asp and Asn), and (v) the remaining linear-chain residues, mainly polar. Ser and Thr have the highest inclination to occur with two different orientations of the side-chain that can be located through crystallography. Such residues exhibiting two chi1 angles have their phi and psi angles in a region that is common to the Ramachandran plots at the two different chi1 angles. The dependence of phi and psi angles on chi1 can be used to understand the helical propensities of some residues. Moreover, the average phi, psi values in the alpha-helices vary with the side-chain conformation.     

Conformational analysis of [Met5]-enkephalin: solvation and ionization considerations

[Met5]-Enkephalin has the sequence Tyr-Gly-Gly-Phe-Met. Only the extended conformation of the peptide has been observed by X-ray crystallography. Nuclear magnetic resonance spectroscopy supports the presence of a turn at Gly 3 and Phe 4 in dimethyl sulfoxide. In this study, the peptide conformational states and thermodynamic properties are understood in terms of ionization state and solvent environment. In the calculation, final conformations obtained from multiple independent Monte Carlo simulated annealing conformational searches are starting points for molecular dynamics simulations. In an aqueous environment given by the use of solvation free energy and the zwitterionic state, dominant structural motifs computed are G-P Type II' bend, G-G Type II' bend, and G-G Type I' bend motifs, in order of increasing free energy. In the calculation of the peptide with neutral N- and C-termini and solvation free energy, the extended conformer dominates (by at least a factor of 2.5), and the conformation of another low free energy conformer superimposes well on the pharmacophoric groups of morphine. Neutralization of charge and solvation induce and stabilize the extended conformation, respectively. A mechanism of inter-conversion between the extended conformer and three bent conformers is supported by phi/psi-scatter plots, and by the conformer relative free energies. An estimate of the entropy change of receptor unbinding is 8.3 cal K-1 mol-1, which gives rise to a -2.5 kcal/mol entropy contribution to the free energy of unbinding at 25 degrees C. The conformational analysis methodology described here should be useful in studies on short peptides and flexible protein surface loops that have important biological implications.     

Molecular dynamics simulations of epidermal growth factor and transforming growth factor-alpha structures in water

AMBER v. 4.1 force field in 1.5 ns NPT molecular dynamics simulations of murine epidermal growth factor (mEGF), human epidermal growth factor (hEGF), and human transforming growth factor-alpha (hTGF-alpha) structures with explicit TIP3P solvation were used to investigate differences in backbone stability, changes in secondary structure, interdomain flexibility, and weakly polar interactions. Backbone root mean square deviations of sections of each peptide show that the most stable regions in mEGF and hEGF are the A-, B-, and C-loops, whereas the most stable regions in hTGF-alpha are the A- and B-loops. The secondary structure in the B-loops of mEGF and hEGF differ significantly from the nuclear magnetic resonance (NMR) structures of mEGF and hEGF. The position and type of turns in the B-loop of mEGF and hEGF increase the interstrand distance of the antiparallel beta-sheets thereby disrupting their structure. The interdomain flexibility of simulated hTGF-alpha structure is greater than in either mEGF or hEGF. The phi, psi dihedrals of hTGF-alpha occupy two distinct populations of phase space corresponding to either a Ceq7 or an alpha-helical conformation. This change in dihedral angle is stabilized by Phe15 with Arg42 and Phe17 with Arg42 N-pi weakly polar interactions that are present only in hTGF-alpha but not in mEGF or hEGF.     

Conformational studies on the selectin and natural killer cell receptor ligands sulfo- and sialyl-lacto-N-fucopentaoses (SuLNFPII and SLNFPII) using NMR spectroscopy and molecular dynamics simulations. Comparisons with the nonacidic parent molecule LNFPII

This investigation is focused on the conformational behavior of the blood group Lewisa (Le(a)-active pentasaccharide lacto-N-fucopentaose II (LNFPII) and its sulfated and sialylated analogs, SuLNFPII and SLNFPII. The latter two are more potent oligosaccharide ligands for the animal lectins, E- and L-selectin, and the natural killer cell receptor, NKR-P1, than are the shorter chain analogs based on the trisaccharide Le(a) domain. We report here that the three oligosaccharides based on the fucopentasaccharide have very similar average solution conformations as determined from NMR spectroscopical parameters, in particular 13C chemical shift differences. From restrained simulated annealing and restrained molecular dynamics (MD) simulations performed in order to determine the most probable conformational distributions around the glycosidic linkages we derive models for these oligosaccharides that are in good agreement with experimental parameters, such as rotating-frame Overhauser effects (ROE's) and long-range 1H,13C coupling constants across the glycosidic linkages. In these model structures the Le(a) domain at the non-reducing end of the longer chain oligosaccharides approximates the same rigid structure as in the shorter analogs. The Gal beta 1-4Glc linkage at the reducing end is also rather rigid, showing only little more flexibility than the Le(a) domain. However, the NeuAc alpha 2-3Gal linkage in SLNFPII, and the GlcNAc beta 1-3Gal linkage in all three oligosaccharides are flexible, in each case fluctuating mainly between two minimum energy structures: (phi = -81 degrees, psi = 8 degrees) and (phi = -160 degrees, psi = -20 degrees) for the NeuAc alpha 2-3Gal linkage, as reported previously for the isomeric sequence 3'-sialyl Le(x), and (phi = -25 degrees, psi = -26 degrees) and (phi = 20 degrees, psi = 24 degrees) for the GlcNAc beta 1-3Gal linkage. The flexibility of the latter linkage may allow the lactosyl domain at the reducing end to fit with little strain into extended carbohydrate binding sites on the recognition proteins, and, for the purposes of drug designs, it will be important to establish which conformational distribution is assumed for the GlcNAc beta 1-3Gal linkage in these longer chain oligosaccharides in the bound state.     

Structure-activity study of the nociceptin(1-13)-NH2 N-terminal tetrapeptide and discovery of a nociceptin receptor antagonist

In the present study, the minimal fragment sequence required to fully activate the nociceptin (NC) receptor, namely NC(1-13)-NH2, was used as template for the design of a series of new compounds. Changes were made in the N-terminal tetrapeptide Phe-Gly-Gly-Phe, which has been shown to be essential for receptor occupation and activation. The new compounds were tested for their ability to inhibit the electrically evoked contraction of the mouse vas deferens, a pharmacological preparation sensitive to NC. Results obtained indicate that (a) the replacement of Gly2 or Gly3 with an aromatic residue (Phe) of L or D chirality eliminates the ability of the peptide to occupy the NC receptor; (b) the distance between Phe1 and Phe4 of NC appears to be critical, since any alteration of it leads to a marked decrease or a total elimination of biological activity; and (c) the insertion of a pseudopeptide bond between Phe1 and Gly2 maintains affinity but eliminates the ability of the peptide to activate the NC receptor and leads to antagonism. The peptide [Phe1psi(CH2-NH)Gly2]-NC(1-13)-NH2 acts as a selective NC receptor antagonist and is inactive on opioid receptors. The results summarized in this paper confirm and extend our previous findings by showing that the structural requirements for NC binding to its receptor are clearly different from those of opioids; in addition, this structure-activity study has led to the identification of the first NC receptor selective antagonist.     

Application of molecular dynamics and free energy perturbation methods to metalloporphyrin-ligand systems II: CO and dioxygen binding to myoglobin

The protein contribution to the relative binding affinity of the ligands CO and O2 toward myoglobin (Mb) has been simulated using free energy perturbation calculations. The tautomers of the His E7 residue are different for the oxymyoglobin (MbO2) and carboxymyoglobin (MbCO) systems. This was modeled by performing two-step calculations that mutate the ligand and mutate the His E7 tautomers in separate steps. Differences in hydrogen bonding to the O2 and CO ligands were incorporated into the model. The O2 complex was calculated to be 2-3 kcal/mol more stable than the corresponding CO complex when compared to the same difference in an isolated heme control. This value agrees well with the experimental value of 2.0 kcal/mol. In qualitative agreement with experiments, the Fe-C-O bond is found to be bent (theta = 159.8 degrees) with a small tilt (theta = 6.2 degrees). The contributions made by each of the 29 residues--within the 9.0-A radius of the iron atom--to the free energy difference are separated into van der Waals and electrostatic contributions; the latter contributions are dominant. Aside from the proximal histidine and the heme group, the residues having the largest difference in free energy in mutating MbO2-->MbCO are His E7, Phe CD1, Phe CD4, Val E11, and Thr E10.     

Synthesis and conformational study of peptides possessing helically arranged delta ZPhe side chains

Z-Dehydrophenylalanine (delta ZPhe) possessing four oligopeptides, Boc-(L-Ala-delta ZPhe-Aib)n-OCH3 (n = 1-4: Boc, t-butoxycarbonyl; Aib, alpha-aminoisobutyric acid), were synthesized, and their solution conformations were investigated by 1H-nmr, ir, uv, and CD spectroscopy and theoretical CD calculation. 1H-nmr (the solvent accessibility of NH groups) and ir studies indicated that all the NH groups except for those belonging to the N-terminal L-Ala-delta ZPhe moiety participate in intramolecular hydrogen bonding in chloroform. This suggests that the peptides n = 2-4 have a 4-->1 hydrogen-bonding pattern characteristic of 3(10)-helical structures. The uv spectra of all these peptides recorded in chloroform and in trimethyl phosphate showed an intense maximum around 276 nm assigned to the delta ZPhe chromophores. The corresponding CD spectra of the peptides n = 2-4 showed exciton couplets with a negative peak at longer wavelengths, whereas that of the peptide n = 1 showed only weak signals. Theoretical CD spectra were calculated for the peptides n = 2-4 of several helical conformations, on the basis of exciton chirality method. This calculation indicated that the three peptides form a helical conformation deviating from the perfect 3(10)-helix that contains three residues per turn, and that their side chains of delta ZPhe residues are arranged regularly along the helix. The center-to-center distance between the nearest phenyl pair(s) was estimated to be approximately 5.5 A. The chemical shifts of the delta ZPhe side-chain protons (H beta and aromatic H) for the peptides n = 2-4 indicated anisotropic shielding effect of neighboring phenyl group (s); the effect also supports a regular arrangement of the delta ZPhe side chains along the helical axis.