A Tryptophan Analog Labeling Method To Study Protein Structure And Function Of Two E. Coli Sulfur Redox Protein Dsba And Dsbe Of The Redox Properties Of The Relationship Between Conformational Change And Function

This dissertation is divided into two parts. Part one (chapter 1) covers studies of protein-protein interaction by using tryptophan analogs. Part two (chapter 2, 3) is about the research on redox properties and conformational changes of DsbE and DsbA proteins and relation to their functions. Tryptophan (Trp) residue has been widely used to study the conformation, intermolecular interaction and dynamics of proteins. However, the presence of several Trp residues makes it difficult to distinguish signals from a mixture of two or more proteins, and limits its utility. Trp in a protein can be replaced by its analogs by biosynthetic incorporation technique. The Trp analogs feature spectroscopic properties that are different from those of the natural Trp residues. In chapter one, we biosynthetically incorporated the tryptophan analogs, 5-hydroxytryptophan and 7-azatryptophan, into IgG binding domain of Streptococcal protein G (PGBD). The exclusive excitation and the novel fluorescence change both in intensity and anisotropy are beneficial to reporting details of the interaction between PGBD and the IgG fragments, and enable assessment of the binding abilities. The dissociation constants are estimated to be 0.28 (M for binding of human Fc and 8.0 (M of mouse Fc, which are in good agreement with the data from other techniques. In part of chapter two, site-directed mutagenesis combined with Trp-analog labeling technique were used to investigate the redox properties and conformational change of DsbA. The results clearly demonstrate that labeling of tryptophan analogs has very little effect on the structure and function and is broadly applicable to quantitatively studying protein-protein interactions in a whole biomolecular complex and local conformational change in a protein.DsbA is the first discovered thiol/disulfide oxidoreductase in the bacterial periplasm. In chapter two, site-directed mutagenesis combined with Trp-analog labeling technique were used to investigate the redox properties and conformational change of DsbA. The results show that: 1) DsbA can catalyze disulfide bond formation efficiently, serving as an oxidase. The reduced form is more stable than the oxidized form; the oxidizing force comes from the tense conformation of the oxidized form. 2) The alteration of local environment around Trp76 between redox forms is responsible for the special fluorescence change. 3) 19F-NMR study provides further evidence that the local conformation of Trp76 is dramatically affected during the redox transformation but that of Trp126 remains unaffected.Escherichia coli DsbE protein (also called as CcmG) is composed of 189 amino acid residues. The hydrophobic N-terminus leads DsbE to traverse cytoplasmic membrane, then to dock onto the membrane with the bulk of the protein faced to the periplasm. DabE contains a thioredoxin domain, which is normally found in thiol/disulfide oxidoreductase (PDI, thioredoxin, DsbA etc). Bacterial genetic experiments suggest that DsbE plays a fundamental role in cytochrome c maturation and that the cysteines of active-site CPTC is essential for cytochrome c maturation. In chapter three, weexpressed and purified leaderless DsbE (DsbEL-), and characterized its structural and functional properties. Our results provide important evidence in understanding this protein. Firstly, DsbE proves a weak reductase; it can catalyze breakage of disulfide bond. The reactivity of the active-site cysteines is relative low (10-4 of that of DsbA). Secondly, during redox reaction, the oxidized DsbE protein undergoes structural transformation resulting in a more stable reduced form. Different redox forms display different spectra of fluorescence, circular dichroism and susceptibilities of tryptophan residues to chemical reagent. We also expressed and purified thioredoxin domain of DsbE protein ((57DsbE). Similar results have been obtained in our research, and the variations between the redox forms are more obvious compared with DsbEL-. Based on our results, we deduce that DsbE protein may maintain the...