In order to investigate the conformational change of the alpha-aminoisobutyric acid (Aib) containing peptide by the D/L replacement of an amino acid residue, single crystals of two diastereomers, Dnp-L-Val-Aib-Gly-L-Leu-pNA (L-L isomer) and Dnp-D-Val-Aib-Gly-L-Leu-pNA (D-L isomer), were prepared from aqueous methanol solutions as CH3OH and CH3OH.H2O solvates, respectively, and were analyzed by the x-ray diffraction method. Molecular conformation of L-L isomer adopts consecutive two different types of beta-turns, a type II' beta-turn bent at Aib-Gly, and a type III beta-turn bent at Gly-Leu, stabilized by two intramolecular (Leu)NH...O = C(Val) and (pNA)NH...O = C(Aib) hydrogen bonds. In contrast, these two intramolecular hydrogen bonds lead the D-L isomer to a distorted 3(10)-helix conformation consisting of consecutive two type-III beta-turn of Aib-Gly-Leu sequence. The most significant structural difference between these diastereomers is the mutual orientation between the Dnp and pNA chromophores. While the extensive stacking of both the chromophores is intramolecularly formed for the folded conformation of L-L isomer, they are oriented toward an opposite direction in the open conformation of D-L isomer and are intermolecularly stacked with each other. The large separation between these diastereomers observed in the chromatography is discussed in the relation with their conformational differences.
We have developed a novel method that effectively identifies the N-terminal product ions produced in the tandem mass spectrometry (MS/MS) analysis of peptides done in conjunction with the specific derivatization of the N-terminal amino group using 5-bromonicotinic acid N-hydroxysuccinimide ester (BrNA-NHS). Electrospray ionization with low-energy collision-induced dissociation (CID) MS/MS clearly differentiated the N-terminal product ions labeled with the 5-bromonicotinyl group from other ions, on the basis of the appearance of CID peaks with a doublet pattern characteristically separated by 2 mass units produced by the equal natural abundances of 79Br and 81Br. The tracing of a series of these bromine-containing product ions allows the easy amino acid sequencing of peptides. Using Gln-Arg-Leu-Gln-Ser-Asn-Gln-Leu-Lys as the test peptide, we found that within 30 minutes at pH 6.5 and 37 degrees C its alpha-amino group was completely acylated with BrNA-NHS (peptide: BrNA-NHS = 1:40; mol/mol). The epsilon-amino group of the C-terminal lysine residue was less likely to be acylated under these conditions, being only partly modified (about 20%). This suggests the possibility of keeping the epsilon-amino group free from acylation. The method was successfully applied to the determination of the amino acid sequences of peptides from porcine kidney aminoacylase I produced by digestion with lysyl endopeptidase and with Staphylococus aureus V8 protease.
Conotoxin GS, a y-carboxyglutamic acid(Gla)-containing neurotoxic peptide composed of 34 amino acid residues with'one Gla residue and three intramolecular disulfide bonds, was synthesized in solution by the Boc strategy, using the cyclohexyl group to protect the y,y-dicarboxyl functional side chain of the Gla residue. All of the protecting groups were removed by the HF procedure. During the synthesis, the Gla residue was completely stable and no decarboxylated product was observed. The free peptide was subjected to the oxidative folding reaction. The reaction proceeded almost quantitatively in the presence of reduced and oxidized glutathione; however, no product was formed in the absence of redox reagents concomitant with the formation of disulfide isomers or intermediates. The final product was confirmed to be identical to natural conotoxin GS on reversed phase-and ion exchange-HPLC as well as capillary zone electrophoresis. The disulfide structure of synthetic conotoxin GS was determined by gas-phase sequencing and mass spectrometry of its proteolytic fragments and was found to be identical to those of other o~-conotoxins. The major disulfide isomer obtained during the oxidative folding reaction without redox reagents was determined in the same manner. To clarify the role of the Gla residue and the disulfide structure in the conotoxin GS molecule, decarboxylated conotoxin GS and its disulfide isomer were also synthesized, and the neurotoxic activities and circular dichroism spectra of these peptides were compared with those of conotoxin GS and its disulfide isomer. The results showed that the correct disulfide structure was necessary for expression of the toxicity; however, the presence of the Gla residue was not a prerequisite for both the activity and the calciumdependent conformational transition.
We have recently undertaken a systematic structural analysis of fully protected tetrapeptides containing at the N- and C-terminus either homo- or heterochiral amino acids, spaced by an achiral dipeptide segment. The interest for this class of peptides derives from the observation that, on reverse-phase (HPLC), the homo- and heterochiral sequences have a markedly different retention times. The diastereomeric sequences, namely Z-(L/D)-Val-X-Y-L-Phe-OMe (X = Sar, Gly, Ac3c, Aib, Ac5c, Ac6c, Deg, Dpg, Dbu, Dip, Dph; Y = Sar, Gly, Ac3c, Aib, Ac5c, Ac6c) show different overall hydrophobicity attributed to a different three-dimensional structure that also depends on the X-Y segment. Therefore, following preliminary studies in solution, we report here the detailed x-ray analysis of the tetrapeptide Z-D-Val-Ac6c-Gly-L-Phe-OMe in order to understand the structural features governing the overall hydrophobicity of linear fully protected tetrapeptides.
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