International journal of peptide and protein research最新文献

It was found that in the case of N-(diisopropylphosphoryl) glutathion (reduced form), 2, N-->S phosphoryl migration took place, but not for N,N-bis(diisopropylphosphoryl) glutathion (oxidized form) or N-diisopropylphosphoryl cysteine. These results were deduced by 31P-NMR tracing experiments. It was shown that phosphoryl migration was catalyzed by an intramolecular carboxyl group, and a mechanism involving a mixed carboxyl-phosphoric anhydride was proposed. A competitive reaction between the amino and thiol group toward diisopropyl phosphite indicated that the phospho-thiol derived from N-(diisopropylphosphoryl) glutathion (reduced form), 2, did not result from direct phosphorylation of the thiol group. N,S-Bis(diisopropylphosphoryl) glutathion provides an authentic sample to confirm the migrated phosphoryl thiol product.

A biotinylated derivative of a designed, difficult-sequence protein (the Minibody, Bianchi, E., Tramontano, A., Sollazzo, M. & Pessi, A. (1993) Int. J. Peptide Protein Res. 41, 385-393) which represented only 3.7% of the crude, cleaved material was quantitatively recovered with about 70% purity, in a single step, by affinity chromatography on immobilised avidin. Purification to homogeneity was then easily achieved by preparative HPLC. This highly effective purification scheme must be contrasted with the previously shown multidimensional, low-yield chromatographic protocol. Since no facilitation of the purification had been obtained by capping alone, this result suggests that capping is useful only in conjunction with affinity chromatography.

Three disulfide linkages of recombinant human brain-derived neurotrophic factor (BDNF) were determined by peptide sequence analysis and characterized by mass spectrometry. The three disulfide bonds for BDNF expressed in Chinese hamster ovary cells include Cys-13-Cys-80, Cys-58-Cys-109 and Cys-68-Cys-111, and the disulfide structure was homologous to that of nerve growth factor.

The synthesis and conformational analysis in solution (by FTIR absorption and 1H NMR) and in the crystal state (by X-ray diffraction) of three Hib-containing depsipeptides have been performed. In the crystal state Z-Aib-Hib-Aib-OMe is folded into a type-III beta-bend, while the conformation adopted by Z-Aib-Hib)2-Aib-OMe is a beta-bend ribbon spiral, characterized by two type-III beta-bends with Aib(1)-Hib(2) and Aib(3)-Hib(4) as corner residues, respectively. Both independent molecules in the asymmetric unit of t-Boc-L-Ala-Hib-L-Ala-OMe crystals are folded into a type-II beta-bend. For the Aib-Hib depsipeptides the conformation adopted in the crystal state is also that largely prevailing in solution, whereas for t-Boc-L-Ala-Hib-L-Ala-OMe the beta-bend conformation is significantly less populated in solution. A comparison is also made with: (i) the published crystal-state conformations of fully protected -(Aib)3-, -(Aib)5-, and -L-Ala-Aib-L-Ala- sequences and the beta-bend ribbon spiral generated by (Aib-L-Pro)n oligomers, and (ii) with the herewith described solution preferred conformation of Z-L-Ala-Aib-L-Ala-OMe. The possible use of Hib as an isosteric replacement for Aib in the design of conformationally constrained depsipeptides is briefly discussed.

The amino acid sequence and chemical interactions at the ends of 163 helices were surveyed so as better to understand amino acid preferences previously observed [Richardson, J.S. & Richardson, D.C. (1988) Science 240, 1648-1652]. Amino acid preferences differed from the previous survey in some significant details and in ways that might affect the choice of amino acids during the design of a protein helix. The following major conclusions about helix ends were deduced from additional patterns of amino acid occurrence and interactions that were observed. (1) A specific pair of hydrogen bonds is often observed between a glutamic acid (or glutamine) side chain at the N3 position and the N-cap amide hydrogen, and between the N-cap side chain (often threonine) and the N3 amide hydrogen. This reciprocal interaction may be an important means of stabilizing the N-terminal end of a helix. (2) Negatively charged amino acids (aspartic acid and glutamic acid) at the N-terminal end of helices may be more important in stabilizing protein helices than positively charged residues (chiefly lysine) at the C-terminal end. (3) The identity of the residue at the N-cap position is correlated with the backbone conformation at that position. (4) Aspartic acid (or asparagine) at the N2 or N3 position may adopt a conformation that suggests a hydrogen-bonding interaction with the end of the helix, especially when the N-cap side chain does not form a hydrogen bond with the end of the helix.

Six model dipeptide methylamides containing dehydroaminobutyric acid (delta Abu) of the type Boc-X-delta z Abu-NHCH3 and Box-X-delta E Abu-NHCH3, X = Ala, Val, Phe (Boc = tert-butoxycarbonyl), have been synthesized and their solution conformations explored using 300 MHz 1H NMR and IR spectroscopy. Studies based on delineation of intramolecularly hydrogen bonded NH groups in CDCl3 and (CD3)2SO revealed that none of the NH groups is appreciably solvent shielded. Difference NOE (Nuclear Overhauser Effect) studies have also failed to detect the presence of any discernible turn structure in these peptides. These studies indicate that the conformational preferences of peptides containing, alpha, beta-dehydroaminobutyric acid are different from those of delta ZPhe and delta ZLeu. It appears that steric interactions due to the beta-substituent in the dehydroamino acid moiety play an important role. Unlike delta ZPhe and delta ZLeu, which have relatively large beta-substituents, phenyl and isopropyl, respectively, and stabilize a beta-turn, the beta-methyl group of delta ZAbu or delta EAbu is readily accommodated in extended conformation. Clearly, the size of beta-substituent in dehydroamino acid crucially influences the conformational preferences. Thus, it may be possible to use different dehydroamino acids to introduce variable but definite constraints in synthetic peptides.

A series of compounds containing a hydroxyethyl-based dipeptide surrogate have been prepared as probes to evaluate the possibility of ICE being an aspartic protease. The aldehyde t-BocAsp(beta-t-butyl)H reacted with the organochromium species derived from phenethyl bromide and CrCl2 to give the expected addition product. Lactonization, reprotection of the amine and oxidation with RuCl3 gave the two protected dipeptide surrogates 7a and 7b. These were incorporated into tetra-, penta- and hexapeptide-like molecules and evaluated as inhibitors of the enzyme. The failure of these compounds to inhibit ICE indicated that this enzyme was very unlikely to be an aspartic protease.

Synthesized beta 1- and beta 2-pentapeptide sequences corresponding to published adrenoceptor transmembrane activation site subtypes were investigated in vitro for selectivity in association for drug ligands of known selectivity. Both nuclear magnetic resonance spectroscopy and molecular mechanics demonstrated that structural differences among the corresponding pentapeptide activation-site sequences can explain agonist selectivity. Results suggest the agonists bind across the activation site loop on the second transmembrane alpha-helix by dipole/dipole interactions between a ligand and the peptide. Since electrostatic interactions within the membrane may determine the rate of intercellular ion flux, agonist association across the activation site sequence could thereby decrease electrostatic resistance to positive ion flux into the cell. Interactions between the peptides and the ligands may provide insight into the structures and mechanisms involved in association of ligands for the identical sequences on the beta-adrenoreceptors.

A series of glycoconjugates, in which [Met5]enkephalin or [D-Ala2,Met5]enkephalin have been linked through an ester bond to the HO-6 of various D-glycopyranose moieties, were synthesized by classical solution methods. The biological activities of these compounds were determined on selective pharmacological preparations: guinea pig ileum and mouse vas deferens for opioid activity, and two mouse cell lines, fibroblasts L929 and melanoma B16BL6, to study the influence on growth processes. The results reported in this study demonstrate the differential effect of the carbohydrate part in enkephalin-related glycoconjugates on receptor recognition. In addition, synthesized neo-glycopeptides stimulate growth of the examined mouse cell lines, whereas parent peptide demonstrated some growth inhibitory properties. Full growth curves showed a dose-dependent effect at concentrations of 10(-7) to 10(-10) M.

The peptide N-Boc-L-Phe-dehydro-Abu-NH-CH3 was synthesized by the usual workup procedure. The crystals grown from methanol at 4 degrees C belong to the space group P2(1)2(1)2(1) with a = 7.589(2), b = 13.690(4), c = 21.897(6) A, Z = 4 and dc = 1.149(5) g cm-3 for C19H29N3O5.CH3OH. The peptide crystals were highly sensitive to radiation. The final agreement factor R was 0.055 for 1109 observed reflections (I > or = 2 sigma) with data extending to a 2 theta value of 103 degrees. The methanol oxygen atom is split into two occupancies. Both sites are involved in identical hydrogen bonding. As a result of substitution of a dehydro-Abu residue at the (i + 2) position the peptide adopts an ideal beta-turn II' conformation with torsion angles of corner residues as phi 1 = 63(1) degrees, psi 1 = -127(1) degrees, phi 2 = -66(1) degrees and psi 2 = -10(1) degrees, and an intramolecular hydrogen bond N-H...O of length 3.01(1) A. This shows that the conformational constraints produced by dehydro-Abu are similar in nature to but different in magnitude than those produced by dehydro-Phe and dehydro-Leu. The methanol-peptide interactions show characteristic features of multiple hydrogen-bond formations involving polar sites of participating peptide and methanol molecules. The packing of the molecules in the unit cell is stabilized by interactions through methanol molecules with the help of several hydrogen bonds.