Conformational analysis of endomorphin-2 by molecular dynamics methods

Endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH(2)) is a highly potent and selective mu-opioid receptor agonist. A conformational analysis of EM2 was carried out by simulated annealing (SA) and molecular dynamics (MD) methods. Molecular modeling was conducted on both neutral (N-terminal NH(2)) and charged (N-terminal NH(3) (+)) molecules. Based on the results of NMR investigations showing an equilibrium mixture of cis and trans Tyr(1)-Pro(2) peptide bonds for EM2 in solution, simulations were performed with restrained cis-Pro and trans-Pro peptide bonds, too. A separate SA study with unrestrained Pro peptide bonds was also conducted. Preferred conformational states are presented in Ramachandran plots. The g(+), g(-), and trans populations of the aromatic amino acid residue side chains were determined in chi(1) space. The distances between the N-terminal N atom and the other backbone N and O atoms, and the distances between the centers of the aromatic rings and the Pro(2) ring, were determined. The energy distribution of the structures obtained by SA was calculated. The preferred secondary structural elements were different kinds of beta-turns, an inverse gamma-turn located in the N-terminal region, and regular and inverse gamma-turns located in the C-terminal region. These turns were stabilized by intramolecular H-bonds and bifurcated H-bonds.     

Morphiceptin and beta-casomorphin-5 analogues containing a reduced peptide bond: selective mu-receptor agonists and a novel mu antagonist, H-Tyr-Pro psi (CH2-NH)Phe-Pro-Gly-OH.

In order to prevent enzymatic degradation of beta-casomorphin-5 (1) and morphiceptin, reduced peptide bonds were incorporated at the 2-3 and 3-4 bonds, respectively. The analogues were synthesized by a combination of solid phase methodology and reductive alkylation of resin-bound peptide amines with Boc-amino acid aldehydes (Boc: tert-butyloxycarbonyl) in the presence of NaBH3CN. During reversed phase high pressure liquid chromatography purification, peak shape distortions could be observed. Epimerization was excluded, based on gas chromatography/mass spectroscopy analysis, which indicated acceptable levels of racemization (less than 3%) in the crude product. Instead, the phenomena could be attributed to slow cis/trans isomerizations originating from the Xxx-Pro bonds in the sequence. The presence of different conformational isomers was also established by 1H-nmr spectroscopy in DMSO-d6. All analogues showed high stability in blood plasma, enhanced binding affinity for the mu receptor, and very low binding to the delta receptor. While the Phe 3 psi(CH2-N)Pro4 analogues (3) and (5) displayed agonist activity, the Pro 2 psi(CH2-NH)Phe3 modified analogue (2) showed antagonist activity comparable to D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2.     

CD-n.m.r. study of the solution conformation of bradykinin analogs containing alpha-aminoisobutyric acid

The conformation in aqueous solution of several alpha-aminoisobutyric acid (AIB)-containing analogs of bradykinin (BK) has been probed by complementary CD and 1H n.m.r. measurements. The conclusion reached is that substitution of AIB for Pro2 and/or Pro3 in BK stabilizes a degree of beta-turn conformation in the N-terminal tetrapeptide moiety of the resulting analogs. Changing the solvent from water to DMSO or TFE further enhances the contribution of particular hydrogen bonded structures to the time-averaged conformation of these peptides. Bradykinin and [AIB7]-BK adopt similar hydrogen bonded conformations in TFE, apparently with a contribution from a beta-turn involving their common Arg1-Pro2-Pro3-Gly4 moiety. The contrasting biological activities of BK and its AIB-analogs are considered in terms of the conformational analogy between the AIB-residue and cis' Pro and the propensity for a beta-turn at the N-terminus of the peptide.     

Assembly of binding loops on aromatic templates as VCAM‐1 mimetics

The design and synthesis of cyclic mimetics of VCAM‐1 protein that reproduce the integrin‐binding domain are presented. The unprotected peptide precursor 37 – 43 , Thr‐Gln‐Ile‐Asp‐Ser‐Pro‐Leu, was grafted onto functional templates of type naphthalene, biphenyl and benzyl through the chemoselective formation of C‐ and N‐terminal oximes resulting in a mixture of four isomeric forms due to syn–anti isomerism of the oxime bonds. Some isomers could be monitored by HPLC and identified by NMR. The molecule containing a naphthalene‐derived template was found to inhibit the VCAM‐1/VLA‐4 interaction more efficiently than previously reported for sulfur‐bridged cyclic peptides containing similar sequences. The finding confirms the importance of incorporating conformational constraints between the terminal ends of the peptide loop 37 – 43 in the design of synthetic inhibitors of the VCAM‐1/integrin interaction. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

How does a symmetric dimer recognize an asymmetric substrate? A substrate complex of HIV-1 protease

The crystal structure of an actual HIV-1 protease-substrate complex is presented at 2.0 A resolution (R-value of 19.7 % (R(free) 23.3 %)) between an inactive variant (D25N) of HIV-1 protease and a long substrate peptide, Lys-Ala-Arg-Val-Leu-Ala-Glu-Ala-Met-Ser, which covers a full binding epitope of capsid(CA)-p2, cleavage site. The substrate peptide is asymmetric in both size and charge distribution. To accommodate this asymmetry the two protease monomers adopt different conformations burying a total of 1038 A(2) of surface area at the protease-substrate interface. The specificity for the CA-p2 substrate peptide is mainly hydrophobic, as most of the hydrogen bonds are made with the backbone of the peptide substrate. Two water molecules bridge the two monomers through the loops Gly49-Gly52 (Gly49'-Gly52') and Pro79'-Val82' (Pro79-Val82). When other complexes are compared, the mobility of these loops is correlated with the content of the P1 and P1' sites. Interdependence of the conformational changes allows the protease to exhibit its wide range of substrate specificity.     

Conformational basis for the biological activity of TOAC-labeled angiotensin II and bradykinin: electron paramagnetic resonance, circular dichroism, and fluorescence studies

N-Terminally and internally labeled analogues of the hormones angiotensin (AII, DRVYIHPF) and bradykinin (BK, RPPGFSPFR) were synthesized containing the paramagnetic amino acid 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). TOAC replaced Asp1 (TOAC1-AII) and Val3 (TOAC3-AII) in AII and was inserted prior to Arg1 (TOAC0-BK) and replacing Pro3 (TOAC3-BK) in BK. The peptide conformational properties were examined as a function of trifluoroethanol (TFE) content and pH. Electron paramagnetic resonance spectra were sensitive to both variables and showed that internally labeled analogues yielded rotational correlation times (tauC) considerably larger than N-terminally labeled ones, evincing the greater freedom of motion of the N-terminus. In TFE, tauC increased due to viscosity effects. Calculation of tau(Cpeptide)/tau(CTOAC) ratios indicated that the peptides acquired more folded conformations. Circular dichroism spectra showed that, except for TOAC1-AII in TFE, the N-terminally labeled analogues displayed a conformational behavior similar to that of the parent peptides. In contrast, under all conditions, the TOAC3 derivatives acquired more restricted conformations. Fluorescence spectra of AII and its derivatives were especially sensitive to the ionization of Tyr4. Fluorescence quenching by the nitroxide moiety was much more pronounced for TOAC3-AII. The conformational behavior of the TOAC derivatives bears excellent correlation with their biological activity, since, while the N-terminally labeled peptides were partially active, their internally labeled counterparts were inactive [Nakaie, C. R., et al., Peptides 2002, 23, 65-70]. The data demonstrate that insertion of TOAC in the middle of the peptide chain induces conformational restrictions that lead to loss of backbone flexibility, not allowing the peptides to acquire their receptor-bound conformation.     

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.     

Energy minimization studies on α-turns

Using a grid search technique, the entire conformational space of a system of four linked peptide units (tetrapeptide) was scanned to pick out geometrically possible 5→1 type hydrogen-bonded conformations defined as an α-turn. The energy minimization of these conformations led to 23 distinct minimum energy conformations (MECs) falling in 13 different classes. The presence of β and γ turn type hydrogen bonds along with 5→1 type hydrogen bond gave conformational variability in a given class. The occurrence of bifurcated hydrogen bonding network was a characteristic feature of most of the MECs. In many prototype MECs non-glycyl residues such as Ala and Pro could be accommodated. Comparison of MECs with the α-turn examples that are observed in proteins showed that the conformationally worked out MECs occurred in isolation in proteins, with the α-helical α-turn being distinctly the most predominant. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational features of bradykinin. A circular dichroism study of some peptide fragments and structural analogues of bradykinin.

The vasoactive hormone bradykinin, its N-and C-terminal fragments and some structural analogues were studied by Circular Dichroism. Conformational features of the peptide can be detected by comparative analysis of the various CD spectra recorded as a function of aqueous pH, solvent and temperature. It is shown that the two biologically essential arginine residues (Arg1 and Arg9) are important for the specific folded bradykinin conformation. Differences between bradykinin, its fragments and analogues become clearly established in conformational terms, and are discussed in relation to the biological activity of these peptides.     

Synthesis and immunosuppressive activity of new cyclolinopeptide A analogs modified with beta-prolines

Immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases. As a continuation of our previous work searching for new, effective suppressors devoid of toxicity, we present the synthesis, conformational analysis, and biological activity of nonapeptides 1-6, analogs of naturally existing immunomodulatory peptide CLA. New CLA analogs were modified with (S)-beta(2)-iso-proline 7 or (S)-beta(3)-homo-proline 8, respectively. The conformational influence of the beta-iso-proline and beta-homo-proline building blocks was analyzed by NMR spectroscopy. Peptides 1-6 exist as a mixture of four isomers due to cis/trans isomerization of the Xxx-Pro peptide bond. The major isomers of peptides 1, 3, and 4 contain all peptide bonds of the trans geometry. The geometry of the proline-proline bond of the second populated isomer of peptides 3 and 4 is cis. The proline-proline peptide bond is cis for the major isomers of peptides 2, 5, and 6. The peptides were tested for their ability to suppress the proliferative response of mouse splenocytes to T- and B-cell mitogens and the secondary humoral immune response to sheep erythrocytes in vitro in parallel with a reference drug-cyclosporine A. The immunoregulatory actions of the peptides depended on the position and content of proline isomers and were, with some exceptions, strongly inhibitory at the highest dose tested (100 microg/ml). In addition, the peptides were practically devoid of toxicity at that dose. In conclusion, the replacement of Pro by beta-Pro may be useful for fine-tuning CLA immunosuppressive potency and undesirable toxicity.     

Purification and properties of a bovine brain thyrotropin-releasing-factor deamidase. A post-proline cleaving enzyme of limited specificity

A bovine brain thyrotropin-releasing-factor (thyroliberin) deamidase has been purified 1100-fold to apparent homogeneity. Molecular weight estimates by gel filtration and sodium dodecylsulfate gel electrophoresis indicate that the enzyme consists of a single polypeptide chain of molecular weight of about 62 000-65 000. The enzyme is inactivated by sulfhydryl blocking agents. Serine proteinase inhibitors, phenylmethanesulfonyl fluoride and benzamidine, have no effect. Besides thyroliberin, the enzyme hydrolyzes peptide bonds involving the carboxyl group of proline residues in luliberin, tuftsin, angiotensin II, melanotropin, and neurotensin. Oxytocin, vasopressin, and bradykinin are not cleaved; they are, however, strong competitive inhibitors of thyroliberin deamidation. The specificity studies indicate that the enzyme is a "post-proline cleaving enzyme" which hydrolyzes peptides of the general structure, Yaa-Pro-Xaa, in which Xaa = amino acid, peptide, or amide (not Pro), and Yaa = N-blocked basic amino acid or a peptide sequence in which the C-terminal residue (i.e. the residue prior to Pro) is a basic amino acid such as His, Lys, or Arg. The enzyme is compared to other post-proline cleaving enzymes.     

The metabolism of neuropeptides. Hydrolysis of peptides by the phosphoramidon-insensitive rat kidney enzyme ''endopeptidase-2'' and by rat microvillar membranes.

Endopeptidase-2, the second endopeptidase in rat kidney brush border [Kenny & Ingram (1987) Biochem. J. 245, 515-524] has been further characterized in regard to its specificity and its contribution to the hydrolysis of peptides by microvillar membrane preparations. The peptide products were identified, after incubating luliberin, substance P, bradykinin and angiotensins I, II and III with the purified enzyme. The bonds hydrolysed were those involving a hydrophobic amino acid residue, but this residue could be located at either the P1 or P1' site. Luliberin was hydrolysed faster than other peptides tested, followed by substance P and bradykinin. Human alpha-atrial natriuretic peptide and the angiotensins were only slowly attacked. Oxytocin and [Arg8]vasopressin were not hydrolysed. No peptide fragments were detected on prolonged incubation with insulin, cytochrome c, ovalbumin and serum albumin. In comparison with pig endopeptidase-24.11 the rates for the susceptible peptides were, with the exception of luliberin, much lower for endopeptidase-2. Indeed, for bradykinin and substance P the ratio kcat./Km was two orders of magnitude lower. Since both endopeptidases are present in rat kidney microvilli, an assessment was made of the relative contributions to the hydrolysis of luliberin, bradykinin and substance P. Only for the first named was endopeptidase-2 the dominant enzyme; for bradykinin it made an equal, and for substance P a minor, contribution.     

Human brain cathepsin H as a neuropeptide and bradykinin metabolizing enzyme

Highly purified human brain cathepsin H (EC 3.4.22.16) was used to study its involvement in degradation of different brain peptides. Its action was determined to be selective. On Leu-enkephalin, dynorphin (1-6), dynorphin (1-13), alpha-neoendorphin, and Lys-bradykinin, it showed a preferential aminopeptidase activity by cleaving off hydrophobic or basic amino acids. It showed no aminopeptidase activity on bradykinin, which has Pro adjacent to its N-terminal amino acid, on neurotensin with blocked N-terminal amino acid, or on dermorphin with second amino acid D-alanine. After prolonged incubation, cathepsin H acted as an endopeptidase. Dermorphin and dynorphin (1-13) were cleaved at bonds with Phe in the P2 position, while dynorphin (1-6), alpha-neoendorphin, bradykinin and Lys-bradykinin were cleaved at bonds with Gly in the P2 position. Further on, it was shown that human brain cathepsin H activity could be controlled in vivo by cystatin C in its full-length form or its [delta1-10] variant, already known to be co-localized in astrocytes, since the Ki values for the inhibition are in the 10(-10) M range.     

Contribution of tertiary amides to the conformational stability of collagen triple helices

The collagen triple helix is composed of three polypeptide strands, each with a sequence of repeating (Xaa-Yaa-Gly) triplets. In these triplets, Xaa and Yaa are often tertiary amides: L-proline (Pro) and 4(R)-hydroxy-L-proline (Hyp). To determine the contribution of tertiary amides to triple-helical stability, Pro and Hyp were replaced in synthetic collagen mimics with a non-natural acyclic tertiary amide: N-methyl-L-alanine (meAla). Replacing a Pro or Hyp residue with meAla decreases triple-helical stability. Ramachandran analysis indicates that meAla residues prefer to adopt straight phi and psi angles that are dissimilar from those of the Pro and Hyp residues in the collagen triple helix. Replacement with meAla decreases triple-helical stability more than does replacement with Ala. All of the peptide bonds in triple-helical collagen are in the trans conformation. Although an Ala residue greatly prefers the trans conformation, a meAla residue exists as a nearly equimolar mixture of trans and cis conformers. These findings indicate that the favorable contribution of Pro and Hyp to the conformational stability of collagen triple helices arises from factors other than their being tertiary amides.     

Raman studies on bradykinin and a cyclic bradykinin

Laser Raman spectra of bradykinin in water, deuterium oxide, and the solid phase were recorded. From the spectra it was concluded that bradykinin conformation is comprised of ordered and unordered structure. The ordered structure appears to be some form of reverse turn. Furthermore, it seems that there is an enhancement of the turn structure in the solid phase. A cyclic cystine containing analog of bradykinin was also examined with Raman spectroscopy. The cyclic bradykinin analog gives a Raman spectrum very similar to that of the linear bradykinin and therefore must share similar conformational forms with bradykinin. The restrictive Cys-Cys disulfide in the cyclic bradykinin must serve to maintain a conformation acceptable to bradykinin receptors since the cyclic peptide exhibits biological activity.     

Enhanced stability of cis Pro-Pro peptide bond in Pro-Pro-Phe sequence motif

Identification of sequence motifs that favor cis peptide bonds in proteins is important for understanding and designing proteins containing turns mediated by cis peptide conformations. From (1)H NMR solution studies on short peptides, we show that the Pro-Pro peptide bond in Pro-Pro-Phe almost equally populates the cis and trans isomers, with the cis isomer stabilized by a CHc...pi interaction involving the terminal Pro and Phe. We also show that Phe is over-represented at sequence positions immediately following cis Pro-Pro motifs in known protein structures. Our results demonstrate that the Pro-Pro cis conformer in Pro-Pro-Phe sequence motifs is as important as the trans conformer, both in short peptides as well as in natively folded proteins.     

Multiple roles of prolyl residues in structure and folding

To explore the ways that proline residues may influence the conformational options of a polypeptide backbone, we have characterized Pro-->Ala mutants of cellular retinoic acid-binding protein I (CRABP I). While all three Xaa-Pro bonds are in the trans conformation in the native protein and the equilibrium stability of each mutant is similar to that of the parent protein, each has distinct effects on folding and unfolding kinetics. The mutation of Pro105 does not alter the kinetics of folding of CRABP I, which indicates that the flexible loop containing this residue is passive in the folding process. By contrast, replacement of Pro85 by Ala abolishes the observable slow phase of folding, revealing that correct configuration of the 84-85 peptide bond is prerequisite to productive folding. Substitution of Pro39 by Ala yields a protein that folds and unfolds more slowly. Removal of the conformational constraint imposed by the proline ring likely raises the transition state barrier by increasing the entropic cost of narrowing the conformational ensemble. Additionally, the Pro-->Ala mutation removes a helix-termination signal that is important for efficient folding to the native state.     

Conformational analysis of the tetrapeptide Pro-D-Phe-Pro-Gly in aqueous solution

Conformational investigations of the tetrapeptide Pro-D-Phe-Pro-Gly in water solution were carried out by 1H and 13C NMR spectroscopy. The internal proline residue allows for the possibility of cis/trans isomerization about the D-Phe-Pro peptide bond resulting in two conformational isomers. The major isomer was identified as the trans isomer. The pH-dependence of the cis/trans equilibrium supports an additional stabilisation of the trans isomer by an intramolecular ionic interaction between the amino- and carboxy-terminus in the zwitterionic state. Based on 13C spin-lattice relaxation times (T1), different pyrrolidine ring conformations of Pro1 and Pro3 could be determined. By combination of several NMR data (vicinal coupling constants 3JN alpha, temperature dependence of the NH chemical shifts, differences in the chemical shifts between the beta and gamma carbons of the proline residues) and energy minimization calculations, a type II' beta-turn should contribute considerably to the overall structure of the trans isomer.