International journal of peptide and protein research最新文献

Hydrolysis of beta A2-casein by bovine chymosin and pepsin A was performed in order to compare the hydrolysis of the two enzymes on this protein. Different conditions have been tested: pH 5.5 for 116h and pH 3.5 for 7 h [E/S = 1/100 (w/w)] for chymosin. pH 3.0 for 24 h [E/S = 1/1000 (w/w)] for pepsin A. Under these conditions 17 peptides were obtained after the action of chymosin and 23 after the action of pepsin A. They corresponded respectively to the cleavage of 14 and 15 peptide bonds for chymosin and pepsin A. However, six of the peptide bonds were only hydrolyzed by chymosin and seven other bonds only by pepsin A. Our results showed a preferential splitting at the Leu-X, Ser-X, and Trp-X bonds for chymosin and Leu-X, Met-X, and Thr-X, for pepsin A. Some of the identified peptides contained sequences with possible physiological roles.

The synthesis of the model compound Aloc-Ala-Ala-Dma-Ala-Ala-OMe has been described as an illustration of the fact that a large group reversibly alkylating the amido group of an oligomer can disturb the regularity of a peptide backbone, oppose its aggregation and thus enhance its solubility greatly, affording synthons for further oligomerization. Application of such a group not only affects the solubility, but alters also the properties of the intermediates. The concomitant change in reactivity may run to such an extent that N-alkylation of oligomers has to be abandoned (this was encountered in the attempted synthesis of Lys-Glu-Dmg). Consequently, the solubility of the growing protected peptide chain will become progressively less and in the mentioned example the oligomerization had to be terminated at the dodecapeptide level, indicating the severe need for reversible "structure-breaking" functions.

We have undertaken a new and more detailed Fourier-transform infrared (FTIR) spectroscopic study of alpha-lactalbumin (in D2O solution) aimed at correlating its secondary structures to observed Amide I' infrared bands. The spectra reported here were interpreted in light of the recently determined crystal structure of alpha-lactalbumin and by comparison with the spectra and structure of the homologous protein lysozyme. Of particular importance is the new evidence supporting the assignment of the band at 1639 cm-1 to 3(10)-helices. This assignment is in excellent agreement with one based on theoretical and experimental studies of 3(10)-helical polypeptides. The frequency observed for 3(10)-helices is distinctly different from that at which alpha-helices are typically found (viz., around 1655 cm-1). In the present study, two bands are clearly resolved in the latter region at 1651 and 1659 cm-1. Both are apparently associated with alpha-helices. These results suggest that for D2O solutions of globular proteins. FTIR spectroscopy can be a facile method for detecting the presence of these two different types of helical conformation and distinguishing between them. This provides a distinct advantage over ultraviolet circular dichroism spectroscopy (UV-CD). This work also provides a basis for future studies of alpha-lactalbumin which examine the effects of environment (e.g., pH, temperature) and ligands (e.g., Ca2+, Mn2+) on its conformation.

The esterification of 2-chlorotrityl chloride resin with Fmoc-amino acids in the presence of DIEA is studied under various conditions. High esterification yields are obtained using 0.6 equiv. Fmoc-amino acid/mmol resin in DCM or DCE, in 25 min, at room temperature. The reaction proceeds without by product formation even in the case of Fmoc-Asn and Fmoc-Gln. The quantitative and easy cleavage of amino acids and peptides from 2-chlorotrityl resin, by using AcOH/TFE/DCM mixtures, is accomplished within 15-60 min at room temperature, while t-butyl type protecting groups remain unaffected. Under these exceptionally mild conditions 2-chlorotrityl cations generated during the cleavage of amino acids and peptides from resin do not attack the nucleophilic side chains of Trp, Met, and Tyr.

MeAla6-cyclosporin A (MeAla6-CsA) is a unique CsA analog that shows weak immunosuppressive activity and yet binds strongly to the proposed cytosolic protein receptor, cyclophilin (CyP). Preliminary 1H NMR data showed significant chemical shift differences between spectra of MeAla6-CsA and CsA, suggesting different preferred conformations. A more detailed study, however, revealed that the backbone conformations of the two molecules are essentially identical, and that the differences can be accounted for, principally, by the sidechain motions of the MeBmt-1, MeLeu-9, and -10 residues. ROE and coupling constant data show that in MeAla6-CsA, the preferred chi 1 rotamers for MeLeu-9 and -10 are + 180 degrees (T), whereas in CsA there is a more even distribution of rotamer populations for MeLeu-10, and a preferred -60 degrees (G-) chi 1 rotamer for MeLeu-9. Similar data argue that the sidechain of MeBmt-1 is more restricted in its motion in MeAla-CsA than in CsA. Temperature studies suggest that these preferred rotamers for MeAla6-CsA may increase the stability of the hydrogen bond between NH(7) and CO(11), but prevent particular residues, especially the essential MeBmt-1 sidechain, from adopting orientations required to elicit immunosuppressive activity. The significant changes observed in the preferred orientations for the sidechains of the MeBmt-1, MeLeu-9, and MeLeu-10 residues in MeAla6-CsA argue that the particular orientations which they assume in CsA are not essential for cyclophilin binding.

Bicyclic peptides are useful model molecules that can mimic the constrained local folding of a great number of natural peptides and proteins, such as ionophoric peptides, enzyme active site, and ligand-receptor active site. The synthesis of the bicyclic title compound with the liquid phase method is described with experimental details. Of particular interest is the heterodetic closure of the second ring. The peptide showed a complexing activity with metal cations like Ba2+, Ca2+, and Mg2+. The free bicyclic peptide conformation in solution has been studied by means of NMR spectroscopy and a plausible structure model worked out with model building on NMR constraints is proposed.

The complete amino acid sequence of recombinant human Cu-Zn superoxide dismutase (CuZnSOD) is presented. The S-carboxymethylated protein was cleaved at lysine residues (with Achromobacter protease I) to provide a set of nine non-overlapping fragments accounting for 90% of the sequence. These fragments were then overlapped and aligned, and the sequence was completed by using peptides generated by cleavage at glutamic acid residues (with S. aureus V8 protease) and at arginine (with clostripain). The recombinant protein contains a single disulfide bond between cysteine residues 57 and 146. The primary sequence of recombinant human CuZnSOD is identical to that predicted by its cDNA sequence.

As core molecule for the multiple attachment of antigenic peptides we have selected the human IgG1 hinge fragment 225-232/225'-232'. Two types of conjugates of this double-chain bis-cystinyl hinge-peptide were prepared i) by linking its C-termini to [NIe15]-human-little-gastrin-[2,17] and ii) by elongating the resulting hinge-peptide/[NIe15]-little-gastrin-[2-17] conjugate at the two N-termini with the human big-gastrin sequence 1-14 to produce the big-gastrin-[1-14]/hinge-peptide/little-gastrin-[2-17] conjugate. For the synthesis of these peptide structures both the route via the preformed double-chain bis-cystinyl peptide and the route via suitably protected monomeric bis-cysteinyl peptides were used. For the latter approach advantage was taken of the previous observation about the preferred oxidation of the bis-cysteinyl hinge-peptide 225-232 to the dimer in parallel alignment. Both synthetic routes led to identical products. Immunization experiments in guinea pigs with the synthetic hybrids led to surprisingly strong immune responses with anti-little-gastrin antibody titers comparable to those induced by the iso-1-cytochrome c/little-gastrin-[2-17] conjugate as carrier-hapten system. These findings show that the two gastrin constructs are fully competent immunogens. Additionally, the gastrin receptor-like specificity of the antibodies indicates that both the synthetic hybrids and the cytochrome c conjugate allow for expression of a little-gastrin-specific conformational epitope similar to the bioactive structure of this hormone. The usefulness of such synthetic hybrids is further confirmed by the observation that the bivalent immunogen, containing both the little-gastrin 2-17 and the big-gastrin 1-14 sequence, is capable of inducing an immune response against both antigenic sequences, although with different efficiency. These results fully confirm our expectations.

Taking advantage of the peptide transport strategy, we have designed and synthesized several new peptides containing 2-aminopimelic acid (Apm), an inhibitor of the diaminopimelate pathway in bacteria: L-Lys-ambo-Apm, ambo-Apm-L-Lys, L-Lys-L-Ala-ambo-Apm, ambo-Apm-L-Ala-L-Lys, L-Ala(Cl)-ambo-Apm and ambo-Apm-L-Ala(Cl). In the two latter cases, Apm was associated with antibacterial amino acid beta-chloro-L-alanine [L-Ala(Cl)], an inhibitor of alanine racemase and transaminase B. The peptides displayed weak or no antibacterial activities; nevertheless, those containing L-Ala(Cl) had low MIC values in the presence of amino acids restoring protein synthesis. When tested on exponential phase Escherichia coli cells grown in minimal medium, the peptides were without effect or bacteriostatic, but important bacteriolytic effects could be observed, especially for the L-Ala(Cl)-containing peptides, when the growth medium was supplemented with specific amino acids. It was demonstrated that the weak or nil effect of the L-lysine-containing peptides was due to a poor uptake.

During the preparation of the NK-2 selective tachykinin antagonist MEN 10208 (Thr-Asp-Tyr-D-Trp-Val-D-Trp-D-Trp-Arg-NH2) and its analogs by the solid-phase method employing the Boc strategy routinely used in our laboratory, we encountered difficulties in the coupling of hydrophobic amino acids D-Trp and Val. To study the coupling problems several syntheses of MEN 10208 and analogs were carried out with different activation strategies. These syntheses yielded considerable amounts of deletion sequences even though a negative Kaiser test was obtained after each coupling. Inaccessibility of the free amino group of the growing peptide due to steric hindrance of the hydrophobic residues during coupling, and for the ninhydrin complex during the Kaiser test, may account, at least in part, for the unsatisfactory synthetics results and for the false-negative ninhydrin tests. Repetition of each synthesis with the Fmoc strategy on a newly developed DOD resin for peptide amides using the DCC/HOBt chemistry gave superior results in terms of the yield and purity of the crude peptides. Therefore, the Fmoc strategy appears to offer advantages over the Boc method for the preparation of these peptides containing hydrophobic amino acids.