Disulfide Bond Influence On Protein Structure And Function&Single-molecule Force Spectroscopy Study On Protein Interactions

Proteins are the material basis of life activities, and widely present in various biological tissues and cells. Disulfide bonds and other interactions between residues provide a basis for protein to maintain the correct structure and function. Whether the Disulfide bonds paired correctly affect the folding of the polypeptide chain. Research of disulfide bonds is very important for protein engineering and artificial drug molecular design. In recent years, mechanical forces was found to play an important role in protein folding. Mechanical force is very common in organisms. Cells and tissues have to interact with external force. With the revolutionary advances in instrument and detection means, single molecule force spectroscopy techniques have developed rapidly. Atomic force microscopy-based single molecule force spectroscopy method has been widely used in the intramolecular and intermolecular interactions, and generates a lot of important research results.In chapter two, we report the fusion expression and characterization of wild and mutant decorsin to study the role of disulfide bonds in protein structure, stability and biological activity. The purified protein shows an obvious inhibition activity to platelet aggregation induced by ADP with IC50of500nM. The remove of cys7-cys15at the N-terminal causes thirty-fold decrease of the inhibition activity with IC50of15μM, whereas the mutation of cys22-cys38at the C-terminal completely impairs the biological activity of decorsin. Meanwhile, the overall secondary and tertiary structures of decorsin are disrupted inevitably without disulfide bonds. Via a domain insertion mutation, the retaining of RGD loop and the adjacent disulfide bond can only warrant very week antihemostatic activity of decorsin. It appears the overall structures of decorsin defined by the conserved cysteine bridges are cooperative for its antihemostatic function. Our study of disulfide bonds together with RGD-sequence is helpful for structure-based drug design on antithrombotic research.In chapter three, we first introduce the background of the ubiquitin and five kinds of domain swapping region. Domain swapping in nearby protein areas happens in the refolding process, which still allows the global structure to keep stable. We obtain genes of five domain swapping protein, existing in refolding process, as well as the natural type of ubiquitin protein by molecular biology approaches. These genes will be expressed in prokaryotic system and further CD results show a general accord with natural ubiquitin protein in their secondary structure. We also apply GB1protein to five domain swapping protein and wild type ubiquitin for (GB1-ubi)4polyprotein, and look into the mechanical stability of these protein by AFM technology. At the end of our experiment, we conclude a high stability of wild type ubiquitin. The stability of remaining protein with domain swapping structure still need more further research.In chapter four, we focus on the interaction in Filamin A by single molecular force spectrum. First we study the unfolding process under external mechanical force of the whole region, and then we measure the unfolding force of every single domain, in order to learn the special structure and biological function of FLNal9-21. There is a difference between the force before and after the interaction of protein and ligand, which will be confirmed in our further study. In addition, we study the FLNa24domain, a significant component in the dimerization of Filamin A, and compare the hydrogen bond in dimers by site-directed mutagenesis and protein cross-linkings. The curves reveal the unfolding process of dimers, but the break of hydrogen bond is unnoticed. We need to apply a new type of protein cross-linkings to the study of hydrogen bond.Based on the results shown above, we have investigated the role of disulfide bonds in protein structure, stability and biological activity. We have a deep understanding of protein interaction by single molecular force spectrum, which helps in the further research of protein structure and functions.