Conformational analysis of biologically active RGD-containing cyclopentapeptides

The theoretical conformational analysis of the biologically active RGD-containing pentapeptide cyclo(-Arg-Gly-Asp-Phe-DVal-), an inhibitor of laminin P1 interaction with its receptor, was performed. The space of permissible torsional angles of the backbone of the molecule was studied by the Monte Carlo method. From the large number of predicted low-energy conformers with various packings of the cyclic moiety of this pentapeptide, only those were selected that corresponded to stable structures of the model linear tripeptide Ac-Ala-Gly-Asp-NHMe. This peptide simulated the spatial possibilities of the backbones of RGD-containing fragments of laminin, vitronectin, and fibronectin. We selected several dozen structures that may be potential biologically active conformers, but only a few of them were capable of forming stable intramolecular hydrogen bonds. We assumed that a biologically active conformer of cyclo(-Arg-Gly-Asp-Phe-DVal-) can be present in significant amounts in an equilibrium mixture in solution along with other conformers without necessarily dominating among them.     

Conformational analysis of the biologically active RGD-containing anti-adhesive peptide cyclo(ArgGlyAspPhe-D-val)

Using theoretical conformational analysis, the RGD-peptide with anti-adhesive activity cyclo(ArgGlyAspPhe-D-Val) was studied. Random sampling was used to search the conformational space of the allowed torsional angles of the main chain of the molecule. Among 900 stable conformers with different folding of the cyclic moiety of the peptide, only those were selected which corresponded to low-energy conformers of the model linear tripeptide AcAlaGlyAspNHMe. This peptide served as the main chain template of the RGD-fragment of the studied cyclopeptide molecule. Of 36 selected cyclopeptide conformers with potential biological activity, only a few contain stable intramolecular hydrogen bonds. It was supposed that a biologically active conformer of the cyclopeptide molecule exists in solution among other conformers, but not necessarily as the major component of the equilibrium mixture.     

Conformational studies of proline-, thiaproline- and dimethylsilaproline-containing diketopiperazines.

As proline plays an important role in biologically active peptides, many analogues of this residue have been developed to modulate the proportion of cis and trans conformers. A correlation between the pyrrolidine ring shape and structural properties of proline has been established. Diketopiperazine (DKP) is the model of choice to study the influence of the proline ring modification. In this contribution, cyclo(Gly-Pro) and two analogues cyclo(Sip-Pro) and cyclo(Thz-Pro) have been studied with proton NMR. We showed that both analogues with heteroatoms in gamma position, silicon and sulfur respectively, display a more rigid five-member ring. The usual flexibility of proline ring is restrained in both cases and only the two C(beta)-exo and C(beta)-endo conformations are observed.     

Conformation of the metastasis-inhibiting laminin pentapeptide

The binding of cancer cells to the basement membrane glycoprotein laminin appears to be a critical step in the metastatic process. This binding can be inhibited competitively by a specific pentapeptide sequence (Tyr-Ile-Gly-Ser-Arg) of the laminin B1 chain, and this peptide can prevent metastasis formationin vivo. However, other similar pentapeptide sequences (e.g., Tyr-Ile-Gly-Ser-Glu) have been found to be much less active in metastasis inhibition, raising the possibility that such amino acid substitutions produce structural changes responsible for altering binding to the laminin receptor. In this study, conformational energy analysis has been used to determine the three-dimensional structures of these peptides. The results indicate that the substitution of Glu for the terminal Arg produces a significant conformational change in the peptide backbone at the middle Gly residue. These results have important implications for the design of drugs that may be useful in preventing metastasis formation and tumor spread.     

Identification of a second active site in laminin for promotion of cell adhesion and migration and inhibition of in vivo melanoma lung colonization

Previously we reported that a pentapeptide (Tyr-Ile-Gly-Ser-Arg or YIGSR) from domain III of the B1 chain of laminin is a cell attachment site with the ability to stimulate cell adhesion and migration and to block experimental metastases. Here we report studies on the activities of synthetic peptides that cover domain III and report a second biologically active peptide PDSGR from this domain with activities similar to YIGSR. We also show that cyclic YIGSR is more potent in these assays than the linear peptide as expected since this sequence on laminin is bracketed by cysteines. Due to their proximity and similar spectrum of activities, it is possible that these sequences act in concert in the native laminin molecule.     

Conformational analysis of the biologically active cyclic analog of beta-casomorphin H-Tyr-cyclo[D-OrnPheProGly].

A biologically active analog of beta-casomorphin, H-Tyr-cyclo[D-OrnPheProGly], was studied by theoretical conformational analysis. Random sampling was used to search the conformational space of allowed dihedral angles. Among 53 low-energy conformers with different folding of the peptide cyclic moiety, only those were selected which correspond to the low-energy area of the model linear tripeptide Ac-D-AlaAlaPro-NHMe. This peptide was used as the main chain "template" for the D-OrnPheProGly fragment of the studied cyclopeptide molecule. Only 15 conformers were chosen as potentially biologically active structures. The conformational possibilities of the Phe residue were constrained to the combined (A + G) region of the Ala residue phi,psi-map for linear peptides.     

The solution conformation of malformin A

Solution conformations of the cyclic pentapeptide plant-hormone malformin A, whose conformational freedom is constrained by an intramolecular disulfide bridge, are derived and presented here. The nmr and CD data of Ptak are used to place restrictions on the search for possible malformin A solution conformers of low energy. Only two distinct conformers were found to be consistent with Ptak's data. Both structures are characterized by an internally buried (solvent-shielded) D -Cys² amide proton, a seven-membered (1–3)hydrogen bond between (N–H) and (O?C), and a disulfide bridge conformation with a P chirality as manifested in the nmr study by the temperature independence of the amide proton chemical shifts for the D -Cys² and D -Leu⁴ residues and the negative sign of the long wavelength maximum in the CD spectrum, respectively. Inspection of space-filling molecular models of both structures indicates severe steric barriers to their rapid interconversion. Thus, it appears that only one of the two conformers may be present in solution. The difference in their calculated dipole moments (4.6 and 6.9D) suggests an experimental method for distinguishing between the two proposed solution structures.     

Solution conformations of some azetidine cyclic peptides

The ¹H-nmr spectra (270 MHz) of cyclic di- and tri-L -azatidine-2-carboxylic acid [cy-clo(L -Aze)₂ and cyclo(L -Aze)³] were determined in CDCl₃ and D₂O and computer simulated. The spectral results were compared with those obtained with cyclo (L -Pro)₂ and cyclo (L, -Pro)₃. In CDCl₃ and D₂O solution, the four membered ring of cyclo (L -Aze)₂ is puckered with the α-proton in a pseudo-axial position, and the ? angle is smaller in absolute value than ?60°, as found for cyclo (L -Pro)_2,. The puckering of the four-membered ring of cyclo(L, -Aze)₃ in CDCl₃ has the α-proton in a pseudo–equatorial position and ? angle larger in absolute value than ?60°, in agreement with cyclo(L -Pro)₃. In D₂O, cyclo(L -Aze)₃ was found to interconvert rapidly between different conformers. In the azetidine cyclic peptides studied, the range of values found for the ? angles was smaller than in the related proline cyclic peptides, indicating greater rigidity in the four-membered ring.     

Cation-binding cyclic peptides with lipophilic tails

We have synthesized and characterized a series of cation-binding cyclic octapeptides which may function as potential ionophoric substances. The materials contain varying degrees of hydrophobic character, which was controlled systematically through the incorporation of N-alkylglycine residues where N-alkyl = methyl, n-hexyl, cyclohexyl, or n-decyl. The peptides reported include cyclo(Phe-Sar-Gly-Sar)₂, cyclo(Glu(OBzl)-Sar-Gly-Sar-Glu(OBzl)-Sar-Gly-(N-decyl)Gly), cyclo(Glu(OBzl)-Sar-Gly-(N-decyl)Gly)₂, cyclo(Glu(OBzl)-Sar-Gly-(N-hexyl)Gly)₂, cyclo(Glu(OBzl)-Sar-Gly-(N-cyclohexyl)Gly)₂, and the corresponding free diacid forms of the Glu-containing compounds. Using ¹³C- and ¹H-nmr spectra, we demonstrated that the mixture of cis/trans peptide bond-isomer conformers, characteristic of the free-peptide benzyl esters in solution, was converted to unique C₂-symmetric, presumably all-trans conformers on complexation with calcium ions. Cation-transport experiments, using the thick-liquid model of transport in a Pressman cell, established that these compounds transport a variety of cations and that one peptide examined in detail, cyclo(Glu(OBzl)-Sar-Gly-(N-decyl)Gly)₂ (selectivity Ca²⁺ > Na⁺ > K⁺ > Mn²⁺ > Cu²⁺ > Mg²⁺ > Co²⁺ > Zn²⁺), transports calcium about an order of magnitude more efficiently than magnesium.     

Organization of intestinal epithelial cells into multicellular structures requires laminin and functional actin microfilaments

Epithelial cell organization into multicellular structures is a critical biological process required for both organogenesis and repair following injury. The basement membrane and the cytoskeleton have important roles in this process; however, the functions of individual components of basement membrane and cytoskeleton are poorly understood. We used IEC-6 cells, a rat intestinal crypt cell line, grown on a three-dimensional gel of reconstituted basement membrane as a model system to determine which extracellular matrix and cytoskeletal components mediate intestinal epithelial cell organization. The cells entered the gel and formed hollow, tubular structures that resembled intestinal crypts. These structures were characterized by a single layer of polarized cells with apical tight junctions and microvilli on the luminal surface. Antiserum to laminin and the pentapeptide Tyr-Ile-Gly-Ser-Arg (which prevents cell attachment to laminin) inhibited this organization, but a control pentapeptide (Tyr-Tyr-Gly-Asp-Ala) and antiserum to collagen IV did not. Cytochalasin B, which interferes with actin microfilament polymerization, also inhibited organization of cells into multicellular structures, but vinblastine and Colcemid, which disrupt microtubules, and cycloheximide, which inhibits protein synthesis, did not. We conclude that organization of intestinal epithelial cells on a basement membrane into multicellular structures results from specific interactions between cells and laminin and requires intact actin microfilaments.     

Hepatocyte attachment to laminin is mediated through multiple receptors

The interaction of hepatocytes with the basement membrane glycoprotein laminin was studied using synthetic peptides derived from laminin sequences. Rat hepatocytes bind to laminin and three different sites within the A and B1 chains of laminin were identified. Active laminin peptides include the PA22-2 peptide (close to the carboxyl end of the long arm in the A chain), the RGD-containing peptide, PA21 (in the short arm of the A chain) and the pentapeptide YIGSR (in the short arm of the B1 chain). PA22-2 was the most potent peptide, whereas the other two peptides had somewhat lower activity. Furthermore, hepatocyte attachment to laminin was inhibited by the three peptides, with PA22-2 being the most active. Various proteins from isolated membranes of cell-surface iodinated hepatocytes bound to a laminin affinity column including three immunologically related binding proteins : Mr = 67,000, 45,000, and 32,000. Several proteins--Mr = 80,000, 55,000, and 38,000-36,000--with a lower affinity for laminin were also identified. Affinity chromatography on peptide columns revealed that the PA22-2 peptide specifically bound the Mr = 80,000, 67,000, 45,000, and 32,000 proteins, the PA21 peptide bound the Mr = 45,000 and 38,000-36,000 proteins and the YIGSR peptide column bound the 38,000-36,000 protein. Antisera to a bacterial fusion protein of the 32-kD laminin-binding protein (LBP-32) reacted strongly with the three laminin-binding proteins, Mr = 67,000, 45,000, and 32,000, showing that they are immunologically related. Immunoperoxidase microscopy studies confirmed that these proteins are present within the plasma membrane of the hepatocyte. The antisera inhibited the adhesion of hepatocytes to hepatocytes to laminin by 30%, supporting the finding that these receptors and others mediate the attachment of hepatocytes to several regions of laminin.     

Identification of the Arg-Gly-Asp sequence in laminin A chain as a latent cell-binding site being exposed in fragment P1

A single RGD-containing sequence present within an epidermal growth factor-like repeat of the short arms of laminin is shown by peptide inhibition to block integrin receptors recognizing a latent cell-binding site of laminin. Based on proteolysis data it is proposed that masking occurs by folding of the globular domain IVa over the cell-binding site in the adjacent rod-like structures of laminin A chain.     

Structure and Polymorphism of the Principal Neutralization Site of Thailand HIV-1 Isolate

Abstract

Refining the geometric parameters for the ensemble of conformers, derived earlier in terms of NMR-spectroscopy data for the immunogenic tip of Thailand HIV-1 isolate, was carried out by quantum chemical methods. As a result, (i) the energy characteristics of initial structures were significanly improved, (ii) their relative locations on the scale of formation heats were determined, and (iii) the energy barriers between conformers under study were computed. On the basis of all data obtained, the high resotion 3D structure model, describing the set of stable conformers and containing the biologically active conformation, was proposed for neutralizing epitope of Thailand HIV-1 isolate. The following major conclusions were made based on the analysis of simulated conformations: i) the Gly-Pro-Gly-Gln-Val-Phe stretch forming the immunogenic crown of Thailand HIV-1 isolate exhibits the properties characteristic for metastable oligopeptide that constitutes in solution the dominant structure with other conformations admissible; (ii) three structures out of five NMR-based starting models form the cluster of conformers which adequately describes general conformational features of this functionally important site of gp120; (iii) two structures residing in this cluster are found to be well-ground for implementing the function of immunoreactive conformation of the stretch of interest; (iv) in spite of this observation, the “global” structure which gives rise to inverse γ-turn in the central Gly-Pro-Gly crest of Thailand HIV-1 gp120 is proposed to be the most probable conformation responsible for the formation of viral antigen-antibody complex in particular case under study.     

Conformational studies on substance P. C-terminal pentapeptide pGlu-Phe-Phe-Gly-Leu-Met NH2

The conformational behavior of the active C-terminal pentapeptide of substance P(SP), pGlu-Phe-Phe-Gly-Leu-Met NH₂ [pGlu-SP(7–11)] was investigated using empirical energy calculations. A sequential approach was used to display the specific contribution of each residue to induce stable conformations of the whole pentapeptide. The most stable conformations include the α_R helix and some partially helical structures; some conformations with glycyl residue in a C₇^eq and C₇^ax configurations (γ and $\text{γ}$ turns) are also favoured. Helical conformations provide a good accessibility of side-chains which play an important role in interacting with the receptor. Fully extended structures and β turns are not specially stable. Such helical stable structures would favour a “lock and key” model of binding.     

Active peptides from the carboxyl-terminal globular domain of laminin α2 and Drosophila α chains

The laminin α1 chain carboxyl-terminal globular domain (G domain) contains multiple biological activities. Recently, we identified five cell binding sequences from the G domain by screening with overlapping 12-mer peptides encompassing the entire domain. The structures of these five sequences in the α1 chain are conserved in the corresponding regions of the different laminin α chains. Here we characterize the adhesion activities of the corresponding peptide segments from both the mouse laminin α2 chain and Drosophila laminin α chain using peptide-coated plastic plates and peptide-conjugated Sepharose beads. Using several cell lines, the laminin α2 chain peptides showed cell attachment and/or spreading activities with cell type specificities. Cell spreading on MG-10 was inhibited by integrin antibodies. Four of the Drosophila laminin peptides showed cell attachment activities. These results suggest that biologically active regions in the G domain are conserved in the laminin α1 and α2 chains, and that these regions in laminin play an important role in cell surface receptor interactions.     

A study of extracellular matrix-cell adhesion peptidic epitopes related to human serum amyloid A (SAA)

Summary Serum amyloid A (SAA), an acute-phase protein, exists normally in the serum while complexed with high-density lipoprotein 3 (SAA-HDL₃). Its levels increase markedly during inflammatory diseases. The pentapeptide Tyr-Ile-Gly-Ser-Asp (YIGSR-like) and the tripeptide Arg-Gly-Asn (RGD-like), related to the cell adhesion domains of laminin and fibronectin, respectively, exist in SAA within close proximity (YIGSDKYFHARGNY; amino acid residues 29–42). A structure-function study of linear and head-to-tail cyclic peptides, related to the amino acid residues 29–42) and 70–76 (GRGAEDS) of human SAA, was performed in order to evaluate their ability to inhibit adhesion of human T-lymphocytes to surfaces coated with extracellular matrix purified from bovine corneal cells.     

Ultraviolet radiation and Vitamin D3 in amphibian health, behaviour, diet and conservation

Amphibians are currently suffering a period of mass extinction with approximately 20% of species under severe threat and more than 120 species already extinct. In light of this crisis there is an urgency to establish viable ex situ populations and also find the causes of in situ declines. The role of ultraviolet radiation and Vitamin D₃ in amphibian health directly influences both ex situ and in situ populations. Vitamin D₃ can be photosynthesised endogenously via UV-B radiation (UV-B), or acquired through the diet, and then metabolised to calcitriol the biologically active hormonal form. Although, there is a lack of literature concerning Vitamin D₃ requirements and calcitriol synthesis in amphibians, amphibians are likely to have similar Vitamin D₃ requirements and metabolic processes as other vertebrates due to the phylogenetically conservative nature of calcitriol biosynthesis. Deficiencies in calcitriol in amphibians result in nutritional metabolic bone disease (NMBD) and could compromise reproduction and immunity. However, excess biologically active UV radiation has also proven detrimental across all three amphibian life stages and therefore could impact both in situ and ex situ populations. Here we review the role and necessity of UV-B and calcitriol in amphibians and the potential for negative impacts due to excessive exposure to UV radiation. We also identify priorities for research that could provide critical information for maintaining healthy in ex situ and in situ populations of amphibians.     

Solution conformation and hydrolytic activity of cyclo(D-leu-L-His)

It has been reported previously that a cyclic dipeptide, cyclo(D -Leu-L -His), showed a high hydrolytic activity toward a hydrophobic ester, p-nitrophenyl laurate. In order to determine the reason for the high catalytic activity, the conformation of cyclo(D -Leu-L -His) in aqueous solution was investigated by nuclear magnetic resonance and circular dichroism spectroscopy and compared with the conformation of cyclo(L -Leu-L -His), which was nearly inactive in otherwise the same conditions for the hydrolysis. It was demonstrated that the spatial arrangement of the hydrophobic isobutyl group of the D -leucyl residue and of the nucleophilic imidazolyl group of the L -histidyl residue in cyclo(D -Leu-L -His) matches very well with the long acyl chain and the active ester function of p-nitrophenyl laurate. On the other hand, in cyclo(L -Leu-L -His) the hydrophobic and the nucleophilic pendant groups are too close with each other to cooperate intramolecularly for the hydrolysis. It was concluded that the different steric structures of the diastereomers can explain the large difference of the catalytic activities.     

Cation binding cyclic peptides composed of imino acid residues

Cyclo(L -Pro-Sar)_n (n = 2–4) with moderate flexibility and hydrophobicity of molecular structure was synthesized, and the characteristics of these cyclic peptides and their metal complexes in acetonitrile were investigated in connection with the residual properties using ¹³C-nmr measurements. The cyclic tetrapeptide cyclo(L -Pro-Sar)₂ showed a sterically hindered phenomenon in acetonitrile in which the amide backbone adopted a cis-trans-cis-trans sequence. The cyclic hexapeptide cyclo(L -Pro-Sar)₃ existed as a mixture of several conformers whose interconversion is slow on the nmr time scale, including cis-cis-trans and/or cis-trans-trans arrangement of the Sar-Pro bond. Finally, it was demonstrated that the cyclic octapeptide cyclo(L -Pro-Sar)₄ behaved as a mixture of multiple conformers which allowed for cis-trans isomerism about the Pro-Sar peptide bond, of which 20–30% had the all-cis Sar-Pro bond isomer and the remaining 70–80% had one (or more) cis Sar-Pro bond isomer. ¹³C-nmr spectra also demonstrated that cyclo(L -Pro-Sar)_n (n = 3,4) formed a 1:1 ion complex whose conformation was characterized by an all-trans peptide bond in the presence of excess metal salt. Cation binding studies, using CD measurements, established that the ion selectivity of cyclo(L -Pro-Sar)₄ in acetonitrile decreased in the order, Ba²⁺ > Ca²⁺ > Na⁺ > Mg²⁺ > Li⁺.     

Theoretical vibrational spectra of cyclohexanecarboxaldehyde

Vibrational spectra of cyclohexanecarboxaldehyde are calculated with density functional theory using the B3LYP functional together with a 6-311++G** basis set and presented. The results in the case of the infrared spectrum of the mixture of conformers at 300 K are compared with the experimental spectrum and, apart from the intensities of the CH and CO stretches, reasonable agreement is found. These deficiencies can be traced back to the well-known nonlinearities in case of CH stretches and the CO stretch. Potential energy distributions among symmetry coordinates in each normal mode are presented and used to assign specific atomic movements to each of the modes. Potential energy scans for the CHO rotor in both the axial and equatorial conformers are presented and barrier heights are compared with previous Hartree–Fock calculations and experimental data. It is reported that there could be three stable conformers, namely equatorial-gauche (eg), which is the most stable, equatorial-trans (et) and axial-gauche (ag). The optimized energies of all the minima and of the transition states are presented. However, comparison of the calculated spectrum with the experimental one indicates that total energies are slightly in error and that in the mixture of conformers no ag is present and thus the et to eg ratio is also different. Using experimental values for relative energies of conformers, we could obtain spectra in fair agreement with experiment. This indicates that when only total energy differences are calculated, slight errors in them play a role because of the very small relative energies in this case, while properties like geometries and spectra, which depend not on energy differences but on analytically calculated energy derivatives, are not affected.Electronic Supplementary Material available.