Rational Design Of Affinity Peptide Ligands And Molecular Mechanism Of Peptide Conformational Transition Affected By Trehalose

This thesis focuses on the application of molecular simulations on protein separation and stability, and affinity peptide ligands and conformational transition of protein structures have been systematically studied.For protein ligand design, a method for peptide ligand design was developed based on docking and molecular dynamics (MD) simulations. The method was demonstrated promising withα-amylase and tissue-type plasminogen activator (t-PA) as model proteins.First of all, based on the molecular structure of the receptor pocket ofα-amylase, pentapeptides were designed and screened by docking simulations. Then, a hexapeptide (FHENWS) was obtained to show high affinity by the simulations. The peptide ligand was immobilized to agarose gel and utilized for the affinity chromatography ofα-amylase. The chromatographic experiments agreed well with the molecular simulations.α-Amylase was then purified by the affinity chromatography from a fermentation broth of Bacillus subtilis, indicating the high affinity and selectivity of the peptide ligand. The method was then extended to a more comprehensive one consisting of amino acid location, docking, molecular surface analysis and MD simulation. This increased the design and screening efficiency. The method led to the discovery of a tetrapeptide of t-PA (QDES). The high binding affinity and specificity of the peptide ligand were confirmed by the purification of t-PA from crude porcine heart extract using the immobilized-ligand column for affinity chromatography.In the simulations of protein structure transformation, the effects of trehalose on protein folding and aggregation have been studied to better understand its effect on protein folding and aggregation.At first, MD simulation of aβ-hairpin peptide at different trehalose concentrations (0-0.26 mol/L) was performed using an all-atom model. It was found that at a proper trehalose concentration (0.065 mol/L), the peptide folded faster than that in water, but it could not fold to theβ-hairpin at higher trehalose concentrations. Free energy landscape analysis indicated that the potential energy barriers in the folding pathway decreased greatly in 0.065 mol/L trehalose, so the peptide folding was facilitated. At higher trehalose concentrations (≥0.098 mol/L), however, trehalose molecules clustered in the peptide region and interacted with the peptide via many hydrogen bonds that prevented the peptide from folding to its native structure. The energy landscape analysis indicated that the potential energy barriers increased so greatly that the peptide could not overcome it, getting trapped in a local free energy basin.All-atom MD simulation was then employed to the study of aggregation behavior of amyloidβ-peptide 16-22 (Aβ16-22, a core sequence of Aβ40 or Aβ42) in the presence of trehalose. Similar with the above observation, the increase of trehalose concentration was found to inhibit the conformational transition of the peptide from the initial coil structure intoβ-strand. That is, at high trehalose concentrations, the Aβ16-22 monomer kept in the secondary structures of turn, bend and coils, which are unfavorable for the intermolecular interactions that lead to aggregation. In this case, there were more hydrogen bonds between trehalose and Aβ16-22, which suppressed the intramolecular hydrogen bond formation in the peptide.The MD simulation was then extended to the study of the conformational transitions of Aβ40 and Aβ42 in the presence of trehalose, and similar results were obtained. The preferential exclusion effect of the peptides on trehalose could suppress hydrophobic interactions, so the hydrophobic collapse of the peptides and the conformational transitions were inhibited.