International journal of peptide and protein research最新文献

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).

Crystals of Eu-(Gly-Gly-Gly).(H2O)5.(ClO4)3 are triclinic, spacegroup P1 with a = 9.123 (2), b = 11.185 (5), c = 11.426 (2) A; alpha = 90.79 (2), beta = 98.08 (1), gamma = 98.57 (2) degrees; Z = 2. The europium cation is surrounded by four oxygens from three different peptide units and four oxygens from water molecules. The geometry around the metal is a distorted bi-capped trigonal prism. The peptide backbone conformation in this complex is compared with those in the free peptide and in various metal complexes. Considerable differences are observed between Eu(III) and Ca(II) complexes of triglycine.

Comparative studies of the hydrolysis of succinyl-Ala2-Phe-methylcoumarylamide with mesentericopeptidase, a mesophilic extracellular serine proteinase from Bacillus mesentericus, and proteinases produced by organisms representing different levels of evolutionary development, were performed. Drastic differences in the proteolytic coefficient kcat/Km were found. As regards their catalytic efficiency, the proteinases studied can be placed in the following order: mesentericopeptidase < subtilisin Novo << subtilisin DY < proteinase K < subtilisin Carlsberg < thermitase < alpha-chymotrypsin. The size of the substrate-binding site of mesentericopeptidase for synthetic peptides was studied by using chloromethyl ketones with the general formula benzyloxycarbonyl-Alan-Phe-CH2Cl (n = 1, 2, 3). The presence of at least five binding subsites (S1 ... S5) on the S-side of the hydrolysed bond was suggested. Studies of the primary specificity of mesentericopeptidase with a series of dipeptide chloromethyl ketones having the general formula benzyloxycarbonyl-Ala-Aa-CH2Cl (Aa = Ala, Val, Leu, Phe) revealed the following order of reactivity toward these inhibitors: Aa = Leu >> Ala > Phe > Val. Kinetically, mesentericopeptidase is similar to subtilisin BPN'/Novo.

The crystal structure determination of three heptapeptides containing alpha-aminoisobutyryl (Aib) residues as a means of helix stabilization provides a high-resolution characterization of 6-->1 hydrogen-bonded conformations, reminiscent of helix-terminating structural features in proteins. The crystal parameters for the three peptides, Boc-Val-Aib-X-Aib-Ala-Aib-Y-OMe, where X and Y are Phe, Leu (I), Leu, Phe (II) and Leu, Leu (III) are: (I) space group P1, Z = 1, a = 9.903 A, b = 10.709 A, c = 11.969 A, alpha = 102.94 degrees, beta = 103.41 degrees, gamma = 92.72 degrees, R = 4.55%; (II) space group P21, Z = 2, a = 10.052 A, b = 17.653 A, c = 13.510 A, beta = 108.45 degrees, R = 4.49%; (III) space group P1, Z = 2 (two independent molecules IIIa and IIIb in the asymmetric unit), a = 10.833 A, b = 13.850 A, c = 16.928 A, alpha = 99.77 degrees, beta = 105.90 degrees, gamma = 90.64 degrees, R = 8.54%. In all cases the helices form 3(10)/alpha-helical (or 3(10)helical) structures, with helical columns formed by head-to-tail hydrogen bonding. The helices assemble in an all-parallel motif in crystals I and III and in an antiparallel motif in II. In the four crystallographically characterized molecules, I, II, IIIa and IIIb, Aib(6) adopts a left-handed helical (hL) conformation with positive phi, psi values, resulting in 6-->1 hydrogen-bond formation between Aib(2) CO and Leu(7)/Phe(7) NH groups. In addition a 4-->1 hydrogen bond is seen between Aib(3) CO and Aib(6) NH groups. This pattern of hydrogen bonding is often observed at the C-terminus of helices proteins, with the terminal pi-type turn being formed by four residues adopting the hRhRhRhL conformation.

The synthesis, physical and analytical characterization, and crystal-state structural analysis by X-ray diffraction of three analogues of the N alpha-acylated tripeptide amide tail of oxytocin, each containing a cyclic C alpha, alpha-disubstituted glycine at position 2, have been performed. The peptides are Boc-L-Pro-Ac3c-Gly-NH2, Z-L-Pro-Ac5c-Gly-NH2 and Z-L-Pro-Ac6c-Gly-NH2. While the former is folded in a type-II beta-turn conformation at the -L-Pro-Ac3c- sequence, the two latter tripeptides form two consecutive (type-II, type-I') beta-turns. The Ac5c- and Ac6c-tripeptides are the first examples of such a highly folded structural combination in a position-2 analogue of the N alpha-acylated -L-Pro-L-Leu-Gly-NH2 sequence.

Conformations of three series of model peptides: homochiral Ac-Pro-L-Xaa-NHCH3 and heterochiral Ac-Pro-D-Xaa-NHCH3 (Xaa = Phe, Val, Leu, Abu, Ala) as well as alpha, beta-dehydro Ac-Pro-delta Xaa-NHCH3 [delta Xaa = (Z)-delta Phe, delta Val,(Z)-delta Leu,(Z)-delta Abu] were investigated by CD spectroscopy in 2% dichloromethane-cyclohexane, trifluoroethanol, water, and occasionally in other solvents. The spectra of homochiral peptides show a significant solvent dependence. Folded structures are present in 2% dichloromethane-cyclohexane and unordered ones occur in water. The folded conformers are of the inverse gamma-turn type for all the peptides but Ac-Pro-L-Phe-NHCH3 for which the type-I beta-turn is preferred. The changes in the spectra of the heterochiral peptides are limited. The compounds adopt the type-II beta-turn in 2% dichloromethane-cyclohexane, represented by class B spectra, and retain this conformation in water as well as in fluorinated alcohols but not always to a full extent. The CD spectra of the unsaturated peptides in 2% dichloromethane-cyclohexane, although they cannot be assigned to any common spectral class, must be attributed to the beta II-turn conformation as determined for these compounds by NMR and IR spectroscopy. The CD spectra of dehydropeptides exhibit a considerable solvent dependence and suggest unordered structures in water.

Recent developments in multiple peptide synthesis have made it feasible to synthesize and screen large numbers of peptide analogs simultaneously. We report here a model study of large scale screening of stereoisomers of substance P with systematic D-amino acid replacements. Such studies are useful in exploring conformational requirements of peptide-receptor interaction and to provide empirical information for peptide drug design. 512 stereoisomers of SP were prepared by the multipin peptide synthesis method. Receptor binding affinities of these analogs were estimated by an iterative competition assay. Results obtained form a comprehensive database on the stereochemical requirement of SP binding to central NK1 receptor. Data from analogs with single D-amino acid replacement are consistent with those previously reported showing that SP binding is highly sensitive to the chirality change of the C-terminal residues (Gln6-Leu10), but less sensitive to the chirality change of the N-terminal residues (Arg1-Gln5). A qualitative analysis of the database by comparison of series of peptide pairs revealed a repeated pattern of affinity change with D-amino acid replacement, suggesting a largely additive binding activity of SP from each residue. On the other hand, possible intramolecular interactions between some N-terminal and C-terminal residues to form an optimal binding conformation were also found. A set of 189 peptides with IC50 values less than 5 microM was subjected to an empirical QSAR analysis using a linear additive model. The relative contribution coefficients obtained agreed with the observation that the predominant contribution comes from the C-terminal residues, suggesting considerable independency of each residue on binding to NK1 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

The multipin peptide synthesis technique, a method for simultaneous multiple peptide synthesis, was developed for large-scale screening of oligopeptides [Geysen et al. (1984) Proc. Natl. Acad. Sci. USA, 81, 3998-4002]. A modification of the technique allows the peptides assembled on polyethylene pins to be cleaved in their native amide form and reconstituted into physiologically compatible solutions. In this study, the suitability of these peptides for quantitative receptor binding assay was evaluated. Substance P and 18 analogs, including a set of N-terminal truncated substance P and a set of naturally occurring substance P analogs, were synthesized by the multipin methods. An average yield of 20 +/- 3 nmol of peptide per pin was obtained. The purity of the peptides was estimated to be ca. 90%. The binding activities of these peptides were determined in a competition assay against 125I-BHSP binding to a rat brain synaptosome preparation. The rank order of the affinities of these peptides depicted a typical pharmacological profile of central NK1 receptor. The IC50 values obtained were also in good agreement with data reported by other groups using similar experimental conditions, except that bulk synthesized peptides were used. This study demonstrates that the peptides synthesized with the multipin technique are suitable for quantitative receptor studies, particularly for a high-volume screening of bioactive peptides.

Systematic energy calculations were performed for a series of LHRH analogs including five agonists with substitutions of D- or N-Me-amino acid residues in positions 4, 6 and 7, and five antagonists with substitutions of D-, N-Me- or alpha-Me-amino acid residues in positions 1, 2, 3, 6, 7 and 10, as well as a bicyclic LHRH antagonist. The geometrical shapes of the calculated low-energy backbone structures for each compound were compared to those of LHRH itself. It appeared that the beta-II' turn at the Tyr5-Gly6-Leu7-Arg8 central tetrapeptide is the common structure for all LHRH agonists considered. LHRH antagonists also possess a common chain reversal in the central tetrapeptide, but it is different from that for LHRH agonists. The LHRH agonists share a similar low-energy conformer at the level of the entire peptide backbone. A characteristic feature of this conformer is a 'surface' formed by a 'polygon' with hydrophobic moieties of pGlu1, Trp3, Tyr5, Leu7 and Pro9 in the corners and with the side chain of the His2 residue in the middle, the latter being crucial for a manifestation of LHRH agonistic activity. Since the N-terminal tripeptide of LHRH presumably participates in a direct interaction with specific receptors, it is legitimate to suggest that the beta-II' turn in the central tetrapeptide maintains the proper spatial arrangement of the N-terminal tripeptide. On the other hand, LHRH antagonists considered in this study were shown to possess low-energy structures, with the spatial arrangement of the residues in the N-terminal tripeptide similar to that of agonists. This would suggest a new approach to the design of LHRH antagonists, namely by stabilizing this specific arrangement, rather than the beta-II' turn in the central tetrapeptide.

Observation of contradictory results with the in vitro assays for inhibin-like activity of the carboxyl terminal 28 amino acid peptide 67-94 with a disulfide loop, of human seminal plasma inhibin (HSPI), prompted us to synthesize both the linear and the cyclic peptides and test their ability to suppress the circulating levels of follicle stimulating hormone (FSH) in vivo in adult male rats. The linear peptide [Cys(Acm)73,87] 67-94 of HSPI was synthesized by solid-phase peptide synthesis using fluorenylmethyloxycarbonyl (Fmoc) chemistry and a continuous-flow technology. The peptide was cyclized by direct iodine oxidation of the S-diacetamidomethyl peptide in dilute solution. In the in vivo assay the linear peptide did not affect the levels of FSH, whereas the cyclic peptide suppressed the levels of FSH significantly. Thus, the carboxyl terminal region of HSPI does have inhibin-like activity and perhaps has the active core of the protein.