Conformation-activity correlations for chemotactic tripeptide analogs incorporating dialkyl residues with linear and cyclic alkyl sidechains at position 2

Five stereochemically constrained analogs of the chemotactic tripeptide incorporating 1-aminocycloalkane-1-carboxylic acid (Ac(n)c) and alpha,alpha-dialkylglycines (Deg, diethylglycine; Dpg, n,n-dipropylglycine and Dbg, n,n-dibutylglycine) at position 2 have been synthesized. NMR studies of peptides For-Met-Xxx-Phe-OMe (Xxx=Ac(7)c, I; Ac(8)c, II; Deg, III; Dpg, IV and Dbg, V; For, formyl) establish that peptides with cycloalkyl residues, I and II, adopt folded beta-turn conformations in CDCl3 and (CD3)2SO. In contrast, analogs with linear alkyl sidechains, III-V, favour fully extended (C5) conformations in solution. Peptides I-V exhibit high activity in inducing beta-glucosaminidase release from rabbit neutrophils, with ED50 values ranging from 1.4-8.0 x 10(-11) M. In human neutrophils the Dxg peptides III-V have ED50 values ranging from 2.3 x 10(-8) to 5.9 x 10(-10) M, with the activity order being V > IV > III. While peptides I-IV are less active than the parent, For-Met-Leu-Phe-OH, in stimulating histamine release from human basophils, the Dbg peptide V is appreciably more potent, suggesting its potential utility as a probe for formyl peptide receptors.     

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.     

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.     

Three-dimensional structure of the ion-channel forming peptide trichorzianin TA VII bound to sodium dodecyl sulfate micelles

Trichorzianin TA VII, Ac0 U1 A2 A3 U4 J5 Q6 U7 U8 U9 S10 L11 U12 P13 V14 U15 I16 Q17 Q18 Fol19, is a nonadecapeptide member of the peptaibol antibiotics biosynthesized by Trichoderma soil fungi, which is characterized by a high proportion of the alpha, alpha-dialkylated amino acids, alpha-aminoisobutyric acid (Aib, U) and isovaline (Iva, J), an acetylated N-terminus and a C-terminal phenylalaninol (Pheol, Fol). The main interest in such peptides stems from their ability to interact with phospholipid bilayers and form voltage-dependent transmembrane channels in planar lipid bilayers. In order to provide insights into the lipid-peptide interaction promoting the voltage gating, the conformational study of TA VII in the presence of perdeuterated sodium dodecyl sulfate (SDS-d25) micelles has been carried out. 1H sequential assignment have been performed with the use of two-dimensional homo- and -heteronuclear nmr techniques including double quantum filtered correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser effect spectroscopy, 1H-13C heteronuclear single quantum correlation, and heteronuclear multiple bond correlation. Conformational parameters, such as 3JNHC alpha H coupling constants, temperature coefficients of amide protons (delta gamma/delta TNH) and quantitative nuclear Overhauser enhancement data, lead to detailed structural information. Ninety-eight three-dimensional structures consistent with the nmr data were generated from 231 interproton distances six phi dihedral angle restraints, using restrained molecular dynamics and energy minimization calculations. The average rms deviation between the 98 refined structures and the energy-minimized average structure is 0.59 A for the backbone atoms. The structure of trichorzianin TA VII associated with SDS micelles, as determined by these methods, is characterized by two right-handed helical segments involving residues 1-8 and 11-19, linked by a beta-turn that leads to an angle about 90 degrees-100 degrees between the two helix axes; residues 18 and 19 at the end of the C-terminal helix exhibit multiple conformations.     

Variability in function measurements of three sensory foot nerves in neuropathic diabetic patients

Although most short, linear peptide fragments of proteins are unstructured in aqueous solution, a number of immunogenic and antigenic peptides have been shown to have conformational preferences for structured forms. By using mainly NMR and CD spectroscopy, it has been possible to detect and quantify quite small populations of beta-turn, helical, and nascent helical conformations. Recent studies have been published indicating that the presence of structured forms is correlated with the location of T cell and/or B cell epitopes in peptide sequences. X-ray crystal structures of complexes between peptides and anti-peptide antibodies frequently show the peptides bound in beta-turn conformations, and the presence of helix in one peptide-antibody complex has been shown by NMR spectroscopy. Studies of peptides free in solution and bound to anti-peptide antibodies in the crystal indicate that the structure of the principal neutralizing determinant of HIV-1 probably includes at least one beta-turn in a highly conserved region. These results can potentially be used in the design of peptide-based vaccines.     

Management of chronic myeloid leukaemia

A tetrasaccharide related to the blood group oligosaccharides, known as sialyl LewisX, has been proposed as the receptor for the lectin responsible for leukocyte adhesion named alternatively as E-selectin or ELAM-1. The 13C- and 1H-nmr spectra have been completely assigned for a tetrasaccharide model of this receptor, Neu5Ac alpha-(2-->3)-Gal beta-(1-->4)-[Fuc alpha-(1-->3)-]GlcNAc beta-NHAc. Quantitative nuclear Overhauser data (NOESY) have been recorded and analyzed by a complete spin matrix simulation method. Conformational space was exhaustively searched and all conformational models whose simulated NOESY spectra matched the experiment were found. Molecular mechanics and molecular dynamics calculations were carried out to test whether the experimental conformations are low energy and thus likely to represent true single conformations for the tetrasaccharide. It was concluded that while the LewisX trisaccharide portion of the compound adopts a single conformation, there is likely to be some flexibility about the Neu5Ac alpha-(2-->3)-linkage. A model featuring fast exchange between two different conformations of this linkage is found to be consistent with both the nmr experiments and the molecular dynamics simulations.     

Design of peptides using alpha,beta-dehydro-residues: synthesis, crystal structure and molecular conformation of Boc-L-Val-delta Phe-delta Phe-L-Val-OCH3

The peptide Boc-L-Val-delta Phe-delta Phe-L-Val-OCH3 was synthesized by the azlactone method in solution phase, and its crystal and molecular structures were determined by x-ray diffraction method. Single crystals were grown by slow evaporation from a methanol/water solution at 6 degrees C. The crystals belong to an orthorhombic space group P212121 with a = 10.478 (6) A, b = 13.953 (I), c = 24.347 (2) and Z = 4. The structure was determined by direct methods and refined by least squares procedure to an R value of 0.052. The structure consists of a peptide and a water molecule. The peptide adopts two overlapping beta-turn conformations of Types II and I' with torsion angles: phi 1 = -54.8 (6) psi 1 = 130.5 (4), phi 2 = 65.8 (5), psi 2 = 12.8 (6), phi 3 = 79.4 (5), psi 3 = 3.9 (7) degrees. The conformation is stabilized by intramolecular hydrogen bonds involving Boc CO and NH of delta Phe3 and CO of Val1 and NH of Val4. The molecules are tightly packed in the unit cell. The crystal structure is stabilized by hydrogen bonds involving NH of delta Phe2 and CO of a symmetry related (x-1/2, 1/2-y, -z) delta Phe2. The solvent-water molecule forms two hydrogen bonds with peptide molecule involving NH of Val1 as an acceptor and another with CO of a symmetry related (1-x, y-1/2, 1/2 -z) delta Phe3 as a donor. These studies indicate that a tetrapeptide with two consecutive delta Phe residues sequenced with valines on both ends adopts two overlapping beta-turns of Types II and I'.     

Conformational analysis of cyclic hexapeptides designed as constrained ligands for the SH2 domain of the p85 subunit of phosphatidylinositol-3-OH kinase

The structures of the cyclic hexapeptide cyclo(-Gly-Tyr-Val-Pro-Met-Leu-) (1) and its phosphotyrosyl (pTyr) derivative cyclo[-Gly-Tyr(PO3H2)-Val-Pro-Met-Leu-] (2), designed as constrained models of a sequence that interacts with the src homology 2 (SH2) region of the p85 subunit of phosphatidylinositol-3-OH kinase (PI-3 kinase), were studied in methanol/water solutions by 500 MHz nmr spectroscopy. Compound 1 was found to exist as a 2:1 mixture of isomers about the Val-Pro bond (trans and cis prolyl) between 292-330 K in 75% CD3O(D,H)/(D,H)2O solutions. A third species of undetermined structure (ca. 5%) was also observed. Compound 2, a model of phosphorylated peptide ligand that binds to the PI-3 kinase SH2 domain, exhibited similar conformational isomerism. When either compound was dissolved in pure solvent [i.e., 100% CD3O(H,D) or (H,D)2O] the ratio of cis to trans isomers was ca 1:1. A battery of one- and two-dimensional nmr experiments at different temperatures and solvent compositions allowed a complete assignment of both the cis and trans forms of 1 and indicated the trans compound to be the major isomer. The spectral properties of the phophorylated derivative 2 paralleled those of 1, indicating like conformations for the two compounds. Analysis of rotating frame Overhauser spectroscopy data, coupling constants, amide proton temperature dependence, and amide proton exchange rates generated a set of constraints that were employed in energy minimization and molecular dynamics calculations using the CHARMM force field. The trans isomer exists with the tyrosine and C-terminal Tyr(+3) (Met) residues at opposite corners of the 18-membered ring separated by a distance of 16-18 A, in contrast with the cis isomer where the side chains of these residues are much closer in space (7-14 A). It was previously shown that the pTyr and the third amino acid C-terminal to this residue are the critical recognition elements for pTyr-peptide binding to the PI-3 kinase SH2 domain. Such cyclic structures may offer appropriate scaffolding for positioning important amino acid side chains of pTyr-containing peptides as a means of increasing their binding affinities to SH2 domains, and in turn provide a conceptual approach toward the design of SH2 domain directed peptidomimetics.     

Physiological functions and regulation of K+ and Cl- channels in single smooth muscle cells

Solution- and solid-state c.d. spectra, as well as surface energetics values, were collected for a series of peptides derived from human salivary proline-rich glycoprotein (PRG). The acronyms and sequences for these peptides are as follows: PRG9-2 = NH2-G(1)-P(2)-CONH2, PRG9-3 = NH2-G(1)-P(2)-P(3)-CONH2, PRG9-4 = NH2-G(1)-P(2)-P(3)-P(4)-CONH2, PRG9-5 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-CONH2, PRG9-6 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-CONH2, PRG9-7 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-CONH2, PRG9-8 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-CONH2, and PRG9-9 = NH2-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9)-CONH2. The presence of stable poly-L-proline II-like 'mini' helices in the solution state was found to be dependent on peptide chain length, pH, salt, and organic solvent type. Other conformational features such as kinks and beta-/gamma-turns were also found in the larger peptides. Solid-state peptide conformations were not necessarily related to their solution-state counterparts. Poly-L-proline II-like 'mini' helices, kinks, and beta-/gamma-turns were similarly found in the various substrate-bound PRG9 peptides. Surface energetics parameters suggested specific orientations for PRG9 peptides and their constituent acids and homopolymers.     

Structural redesign and stabilization of the overlapping tandem beta-turns of RNA polymerase II

Peptides representing single repeat units of the carboxy-terminal domain (CTD) of RNA polymerase II (Tyr-Ser-Pro-Thr-Ser-Pro-Ser-Tyr-NH2, 1) contain overlapping Ser-Pro-Xaa-Xaa beta-turn forming sites which permit their overall structure to closely resemble members of the quinoxaline class of antitumor DNA bisintercalators. We have modified this native sequence at the i+2 positions of each beta-turn unit by substituting Gly or D-Ala in an attempt to preorganize this structure in aqueous solution. CD and NMR spectroscopic investigations confirmed the presence of type II beta-turns within each of the substituted peptides in contrast to the native sequence which contains a relatively low population of turn structure. In addition, an examination of singly substituted peptides suggests that an increase in the population of beta-turn structure within the amino-terminal Ser-Pro-Xaa-Xaa site also increased the formation of beta-turn structure in the carboxy-terminal (unmodified) Ser-Pro-Xaa-Xaa site; in comparison, substitution in the carboxy-terminal site did not influence structure in the remaining portion of the peptide. Overall, these results suggest that the structures formed could provide unique, preorganized linkers for the construction of novel DNA-interactive bisintercalators.     

NMR solution structure of the receptor binding domain of Pseudomonas aeruginosa pilin strain P1. Identification of a beta-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 [McInnes, 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 beta-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 beta-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.     

Pegylated peptides. IV. Enhanced biological activity of site-directed pegylated GRF analogs

Conditions have been developed for the site-specific pegylation (NH2-terminus, side-chain and carboxy-terminus) of a potent analog of growth hormone-releasing factor, [Ala15]-hGRF(1-29)-NH2. These pegylated peptides were prepared by solid-phase peptide synthesis using the Fmoc/tBu strategy, and were fully characterized by analytical HPLC, amino-acid analysis, 1H-NMR spectroscopy and laser desorption mass spectrometry. Biological activities of hGRF analogs were determined in vitro utilizing stimulation of growth hormone release by cultured rat pituitary cells as an index. GH-releasing potencies of the pegylated hGRF analogs were compared to a series of model analogs of [Ala15]-hGRF(1-29)-NH2 that were acetylated or protected as the ethylamides at the pegylation sites. It was found that acetylation at the NH2-terminus resulted in reduced potency, which was not further affected when the NH2-terminus was pegylated, regardless of the size of poly(ethyleneglycol) (PEG) employed (e.g. PEG2000 or PEG5000). Pegylation at Asp8 or Lys12 decreased biological potency, a situation which was exacerbated by increasing the molecular weight of PEG. Pegylation at Lys21 or Asp25 did not significantly affect biological activity. The C-terminal model peptide, [Ala15,Orn(Ac)30]-hGRF(1-29)-NH2, was the most potent analog identified in this series (ca. 4-5-fold that of hGRF(1-44)-NH2. The COOH-terminal pegylated analogs retained this increased level of biological activity independent of PEG molecular weight. These studies demonstrate that a biologically active peptide can be pegylated and retain the full in vitro potency of the peptide. However, the biological activity is highly dependent on the site of pegylation and, in some cases, the molecular weight of PEG (degree of pegylation) moiety used.     

Structure-activity relation of NH2-terminal human parathyroid hormone fragments

Human parathyroid hormone (hPTH) is involved in the regulation of the calcium level in blood. This hormone function is located in the NH2-terminal 34 amino acids of the 84-amino acid peptide hormone and is transduced via the adenylate cyclase and the phosphatidylinositol signaling pathways. It is well known that truncation of the two NH2-terminal amino acids of the hormone leads to complete loss of in vivo normocalcemic function. To correlate loss of calcium level regulatory activity after stepwise NH2-terminal truncation and solution structure, we studied the conformations of fragments hPTH-(2-37), hPTH-(3-37), and hPTH-(4-37) in comparison to hPTH-(1-37) in aqueous buffer solution under near physiological conditions by circular dichroism spectroscopy, two-dimensional nuclear magnetic resonance spectroscopy, and restrained molecular dynamics calculations. All peptides show helical structures and hydrophobic interactions between Leu-15 and Trp-23 that lead to a defined loop region from His-14 to Ser-17. A COOH-terminal helix from Met-18 to at least Leu-28 was found for all peptides. The helical structure in the NH2-terminal part of the peptides was lost in parallel with the NH2-terminal truncation and can be correlated with the loss of calcium regulatory activity.     

Structural characteristics for biological activity of heat-stable enterotoxin produced by enterotoxigenic Escherichia coli: X-ray crystallography of weakly toxic and nontoxic analogs

Heat-stable enterotoxin (ST) produced by a pathogenic strain of Escherichia coli exerts its function by binding to a membrane-bound guanylyl cyclase on intestinal epithelial cell membranes, which in turn catalyzes the production of cyclic GMP as a second messenger in the cells. To elucidate the structural requirements for the biological activities of ST, we synthesized [Mpr5,Gly13]STp(5-17) and [Mpr5,Leu13]STp(5-17), which are weakly toxic and nontoxic analogs of STp, in which the toxic domain consists of the sequence from Cys at position 5 to Cys at position 17. In these analogs, Cys at position 5 is replaced by Mpr (beta-mercaptopropionic acid) and Ala at position 13 by Gly and Leu, respectively. We examined these analogs by X-ray diffraction analysis using direct methods and refined the structures to crystallographic R factors of 7.3% and 6.6% using 5492 and 5122 data, respectively, observed > 3 sigma (Fo) with a resolution of 0.89 A. These peptides have a right-handed spiral structure consisting of three structural segments: an N-terminal 3(10) helix, a central type I beta-turn, and a C-terminal type II beta-turn. These structures show minor differences from that of [Mpr5]STp(5-17), the fully toxic analog of heat-stable enterotoxin [Ozaki et al. (1991) J. Biol. Chem. 266, 5934-5941], suggesting that the decrease and loss of the biological activities of [Mpr5,Gly13]STp(5-17) and [Mpr5,Leu13]STp(5-17), respectively, are not caused by structural changes but are associated with the direct interaction of Ala13 with the receptor protein. Careful comparison of these structures in crystalline states revealed that ST has the following structural characteristics: (i) inherent flexibility at the junctions of the three segments and in the central segment, which includes the putative receptor-binding residues, Ala13, (ii) a specific hydrophobic character around the central segment, and (iii) an unexpected C-terminal folding similar to those of functionally unrelated peptides that are known to be ionophores.     

CD of proline-rich polypeptides: application to the study of the repetitive domain of maize glutelin-2

An overview of CD of proline-rich peptides is reported. First, structural characteristics, theoretical CD studies, and the biological relevance of polyproline II structure in such peptides are discussed. Second, a CD study of peptides belonging to the repetitive domain of maize glutelin-2, H-(Val-His-Leu-Pro-Pro-Pro)n-OH (n = 3, 5, 8), is described. This series of peptides displayed the CD features of polyproline II structure in water (5 degrees C, pH 5). Moreover, it was shown that the addition of increasing amounts of the polyanionic molecule heparin forced a displacement of the conformational equilibrium of those peptides toward higher proportions of the polyproline II structure. In contrast, when the temperature is raised such a structure gradually disappears, leading to more disordered conformations.     

Role of beta-turn residues in beta-hairpin formation and stability in designed peptides

The sequence RGITVNGKTYGR has been reported as part of a de novo design peptide system. This peptide folds as a beta-hairpin structure with three residues per strand and two residue turns. Asn6 side-chain, the residue in position L1 of the beta-turn, appeared to be solvent exposed, interacting only within the turn but not with the rest of the peptide. We have chosen this position as a good candidate to design mutations, based on the protein database statistical abundances, that should mainly affect the turn stability and possibly the pairing between strands. We have found that all NMR parameters, in particular the conformational shift analysis of CalphaH and the coupling constants, 3JHNalpha, correlate very well and show similar conformational features in all the turn mutant peptides. The population estimates are in reasonable agreement among the different methods used. It appears that the peptide with Asn in position L1 is the most structured peptide, followed by the one with Asp6. The next structured peptide is the one with Gly6. The least populated peptides were those with Ala6 and Ser6. We have found a strong correlation between the hairpin population, as determined from the conformational shift of CalphaH and the occurrence of the different residues at position L1 of beta-hairpins with type I' beta-turn, in the protein database. Our analysis demonstrates that this peptide system is sensitive enough to register small energy changes in the hairpin structure; therefore, it constitutes an appropriate model to quantify energy contributions, once the appropriate sheet/coil transition algorithm is developed. Comparison with the other studies indicate that the design of a specific hairpin structure must involve a sequence at the turn region favouring the desired turn type, and a sequence at the strands that avoids alternative interstrand side-chain pairings.     

An NMR study on the DNA-binding SPKK motif and a model for its interaction with DNA

The solution structure of one and two repeats of the 'SPKK' DNA-binding motif is reported on the basis of NMR measurements. In dimethylsulphoxide (DMSO) the major population (approximately 90%) of peptides, SPRKSPRK(S2) and GSPKKSPRK(S2b), adopts a conformation, which has two trans prolines. The two 'SP(R/K)K' units in these peptides are equivalent and each adopts a turn structure exchanging with an extended structure. This is suggested by an NOE connectivity of the beta-turn type, between the backbone amide protons of residues (i+2) and (i+3) and NOE connectivities of the Asx(sigma)-turn type, between protons of the ith Ser and the backbone amide proton on residue (i+2). This suggests that each SP(R/K)K unit has a structural intermediate between (or a combination of) a beta-turn and an Asx(sigma)-turn. In 90-10% DMSO/H2O at 4 degrees C the two units of S2 are connected more tightly by folding into a short 3(10) helix, broken at the second proline. For another peptide, Thr-Pro-Arg-Lys(T1), the major population (75%) in 100% DMSO comprises a beta-turn in rapid exchange with an extended structure. We did not observe an NOE connectivity of the Asx(sigma) type with the T1 peptide. A possible structure of the SPKK motif in the complex with DNA is discussed.     

The MHC of a broiler chicken line: serology, B-G genotypes, and B-F/B-LB sequences

The tumor suppresser protein p53 has been called the "guardian of the genome." DNA damage induces p53 to either halt the cell cycle, allowing for repair, or initiate apoptosis. P53 is mutated in over 50% of human tumors and it has been proposed that many tumorigenic mutations are deleterious to p53 because they induce local unfolding. To explore this hypothesis, peptide models have been developed to study tumorigenic mutations in the H2 helix of the p53 core domain. This helix is rich with charged residues and is a key component of the DNA binding region. A 16-residue peptide corresponding to the H2 wild-type sequence extended with an Ala-rich C-terminus was synthesized and studied by 1H-nmr (500 MHz) and CD. The nmr studies demonstrate that this peptide adopts helical structure in solution. Six additional peptides corresponding to subtle tumorigenic mutations were synthesized and CD was used to assess the relative stability of these "mutant analogues." All six mutations studied are destabilizing relative to the wild type, with delta delta G values in the range of 0.26 to 1.35 kcal mol-1. Surprisingly, substitution of Asp 281 with Ala resulted in a peptide with the greatest destabilization even though Ala possesses the largest helix propensity of the common 20 amino acids. Because this helix appears to be stabilized mainly by local electrostatics, we conclude that its structure is susceptible to even the most conservative mutations. These results provide support for the hypothesis that tumorigenic mutations induce local unfolding of p53.     

The crystal state conformation of Aib-rich segments of peptaibol antibiotics

Ac-(Aib-Ala)3-OH (a protected segment of the peptaibols gliodeliquescin and paracelsin), Z-Leu-Aib-Val-Aib-Gly-OtBu (a segment of [Leu]7-gliodeliquescin), Z-Val-Aib-Aib-Gln-OtBu (a common segment of alamethicin, paracelsin, and hypelcin), and Ac-Aib-Pro-(Aib-Ala)2-OMe and Z-Aib-Pro-(Aib-Ala)2-OMe, which represent differently N(alpha)-protected 1-6 segments of alamethicin and hypelcin, have been synthesized by solution methods. The crystal-state conformations of these five Aib-containing peptides have been determined by X-ray diffraction analysis. We have confirmed that the 3(10)-helical structure is preferentially adopted by Aib-rich short peptides. An experimentally unambiguous proof for the 3(10)-->alpha-helix conversion has been provided by the two differently N-blocked -Aib-Pro-(Aib-Ala)2-OMe hexapeptides. The beta-bend ribbon conformation, commonly observed in the (Aib-Pro)n sequential oligopeptides, is not found in the -Aib-Pro-Aib-Ala-Aib-Ala-sequence. As expected on the basis of the L-configuration of the C(alpha)-monoalkylated residues, a right-handed helix screw sense was found in all peptides investigated.     

Glycodermorphins: opioid peptides with potent and prolonged analgesic activity and enhanced blood-brain barrier penetration

1. In order to improve the in vivo stability of the opioid peptide dermorphin we synthesized O-betaglucosylated analogs ([Ser7-O-betaGlc]dermorphin and [Ser7-O-betaGlc(Ac)4]-dermorphin) and C-alphagalactosylated analogs ([Ala7-C-alphaGal]dermorphin and [Ala7-C-alphaGal(Ac)4]-dermorphin). 2. O- and C-glycosylation of dermorphin halved the peptide affinity for brain mu-opioid receptors and the biological potency in guinea-pig ileum assay (GPI). Despite their lower opioid receptor affinity, when administered intracerebroventricularly (i.c.v., 8-40 pmol) and subcutaneously (s.c., 0.5-3 micromol kg(-1)) in rats, glycosylated analogs were two times more potent than dermorphin in reducing the nociceptive response to radiant heat. Acetylation of sugar hydroxyl groups reduces 5-10 times both biological activity on GPI and mu-receptor affinity, whereas the antinociceptive potency was equal to (i.c.v.) or only two-three times lower (s.c.) than dermorphin potency. 3. Blood-Brain Barrier Permeability Index (BBB-PI) of the glycodermorphins was significantly higher than that of dermorphin, indicating a facilitated entry into the brain: O-beta-linked glucoconiugates are expected to enter CNS by the glucose transporter GLUT-1 of the endothelial barrier. However the calculated BBB-PI for the C-alphagalactoside was about two times higher than that of the O-betaglucoside, excluding the implication of GLUT-1 that is known to be selective for O-beta-links and preferring for the exose glucose. 4. The enhanced brain permeability with the subsequent decrease in peripheral dosage of these opioid peptides did not result in lowering constipation.