Studies On The Molecular Design Of Antiviral Peptide GBVA10 And The Chromatography Methodology Of Peptides

1 Studies on the molecular design of antiviral peptide GBVA10Background:The family Flaviviridae comprises three genera. They are Hepacivirus, Flavirus and Pestivirus. Hepatitis C virus (HCV) is the sole member of the genus Hepacivirus.GB virus is one member of the genus Pestivirus and shows much homology with Hepacivirus in the view of evolution. NS5 A, a non-structure protein of HCV, includes a segment of helix sequence. The helix is an amphipathic structure, can anchor on membrane, and lead to the association between mernrane and virus particles. Studies prove the anti-HCV activities of the N-terminal 1-31 amino acids in this sequence. Since the high homology of GB virus and HCV studies on the N-terminal of HCV non-structure protein (NS5A) membrane anchor domain become more interesting. During the previous work of this study, the a-helix sequence is the template of the N-terminal of GBV-A NS5A membrane anchor domain. The basic sequence of 24 amino acids of the N-terminal was chosen and named as GBVA-1 (DLLDCWVRLGRYLLRRLKTPFTRL). First, the sequence was truncated from both terminus; second, amino acid residue shifts were made through the sequence in a sequence length of 18 amino acid residues. Showing the highest anti-HCV activity GBVA10 was obtained from all designed peptides via HCV infection assay. The sequence of GBVA10 is CWVRLGRYLLRRLKTPFT.Objectives:In this thesis, GBVA10 is a template peptide. We substituted some amino acids in purpose to do rational molecular design of the peptide sequence. Anti-HCV peptide sequence of potential application values is expected to be flitrated via the alterations of hydrophobic, helicity and electric charge.Results:In RP-HPLC, retention time of peptide is the measurement of hydrophobic property, hydrophobicity of peptides is related with the amion acid constitution, primary sequence character and hydrophobic interactions within amino acid sequence. Almost all the peptides of GBVA10 groups include cysteine, therefore disulfide bond is easy to form when there is DMSO in existence. Thus there is no DMSO used in the experiments. However, the role of Cys in the biological activity of peptide needs further study.The secondary structure of GBVA10 peptides is a-helical structure, there is correlation between helicity and hydrophobicity of peptides. The hemolysis of peptide shows strong relationship with hydrophobicity and electric charge. Reduction of hydrophobicity and electric charge significantly decreased the hemolytic activity. The result indicates that the series of GBVA10 peptides probably destroy the erythrocyte via hydrophobic interactions and charge absorption effects. Peptides Y8E/T15E, C1E/Y8E/T15E and C1E/Y8E/T15E/T18E in electric charge groups need the peptide concentration over 500ug/ml to show significant hemolysis. However, their hydrophobic property is similar with the hydrophobic property of the parent peptide GBVA10. Y8E/T15E, C1E/Y8E/T15E have more helicity than the parent peptide, C1E/Y8E/T15E/T18E is only a little lower. We can see that their hydrophobic property and secondary structure are almost no significantly demolished after the molecular design, but hemolytic activity reduces significantly. Therefore, Y8E/T15E, C1E/Y8E/T15E and C1E/Y8E/T15E/T18E are the anti-HCV peptide with potential application values for further research.2 Chromatography methodology of peptidesBackground:RP-HPLC is based on the theory that,the hydrophobicity of solid phase is higher than the mobile phase to separate the samples and the samples combine with the solide phase due to the hydrophobic interaction. Through the adjustment of organic solvent gradient in the mobile phase, the samples of difference hydrophobicity release from the solid phase. RP-HPLC shows more and more applications in the fields of isolation, purification and identification of protein, peptide and nucleic acid. According to the literatures, amphipathicα-helix peptides in RP-HPLC exhibit self-association ability, besides the adsorption-desorption equilibrium with the solid phase. Hypothesis 1, at low temperature, peptides associate through its hydrophobic face to become dimmers. Hypothesis 2, at higher temperatures, peptides unfold into monomeric form or random coil form. Peptides are always bound in its monomeric form to the solid phase of RP-HPLC.Objectives:The two chiral centers in the molecule of Ile, one of which at theα-site of the amino acid and the other one at the side chains of the amino acid, could produce four various chiral stereo-isomers. Utilizing RP-HPLC, we compared the isomers of Ile and the polypeptides containing the Ile isomers and showed that the Ile in peptides exhibited different retention times during RP-HPLC. Through RP-HPLC, we studied the interaction of samples and stationary phase and the sample-sample interaction, showing that RP-HPLC is a sensitive method to separate peptides with amino acid stereo-isomers.Results:Samples are separated by RP-HPLC, based on the hydrophobic interactions between sample and stationary phase composed of silicon substrate and alkyl chains. The separation of different samples could be determined in different retention time. Chiral isomers of the amino acid without the secondary structure cannot be separated resulting in the same retention time. For the mixture of the chiral isomers of amino acid with similar characters, RP-HPLC has a characteristics of co-elute. RP-HPLC is very sensitive to the chiral isomers of the polypeptides molecule. As a result, compared to the amino acid residues without the secondary structure, the RP-HPLC resolution of the polypeptides containing chiral-isomer amino acids is much higher, and there is large difference in retention time and apparent chromatographic peaks. When the temperature increases, mobile phase viscosity decreases and the adsorption-desorption effect between sample and stationary phase become more active, leading to the earlier retention time and the increase of the resolution. In the same temperature conditions of RP-HPLC, the retention times of single analysis and mixed analysis of polypeptides molecule are different. The difference in retention time of single analysis is large but of mixed analysis is very small, indicating that the interaction between various polypeptides molecules exists in addition to the interaction between polypeptides molecules and the stationary phase. This interaction appears in the continuous adsorption-desorption process, leading to the shorter retention time of polypeptides. On the contrary, the retention time of polypeptides becomes greater than the the original retention time.