Development of Molecular Tools and High Throughput Screens in Protein Engineering
Lim, Kok Hong Sean
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This dissertation represents an effort of to utilize various protein engineering approaches and methods, together with the understanding of the basic of protein biochemistry, to design novel molecular tools and high throughput screening methods for the study and characterization of the interactions between proteins, a protein and a ligand, as well as a protein and a peptide. The stability and activity of streptavidin depend upon the oligomerization of the four identical subunits. Therefore, disruption to subunit association through mutations at the subunit interface compromises both the stability and activity of the protein. To restore the function and stability of protein upon subunit dissociation, we described a systematic strategy for the construction of streptavidin monomers and dimers. The mutations that were rationally engineered by minimizing binding pocket flexibility and stabilizing originally buried native dimer interface through the introduction of disulfide bond and salt bridges on subunit interface, have resulted in substantial improvement in both binding affinity and thermostability of the protein, compared to previously engineered mutants that lack these stabilizing mutations. These engineered streptavidin monomer and dimer mutants were shown to label biotinylated receptors on the cell surface efficiently, which demonstrates their applicability in the field of molecular detection. To further improve the binding affinity and thermalstability of the protein, we adopted protein homology modeling approach to create a hybrid of two avidin like proteins, streptavidin and rhizavidin. The resulting hybrid, named as mStrav, has greatly improved binding affinity by approximately 44-fold and thermalstability by approximately 28 °C. We showed that mStrav (1) can be used as a detection tool for recognizing surface immobilized biotinylated ligand, (2) can be expressed stably on yeast surface and mammalian surface to trap biotinylated ligands, and (3) can be fused to fluorescence proteins (EGFP and YPet) to create bifunctional molecules capable of monovalent biotin detection. Such versatile streptavidin monomer, mStrav, should be a useful reagent for designing novel detection systems based on biotin recognition. Obtaining a molecular description of protein-protein interactions (PPI) is important to build an accurate model of structure-function relationship. Detailed models of PPI can also facilitate the development of novel molecular reagents to regulate key biological processes. The formation of protein complexes is critical for carrying out biological functions, hence, it is useful to design a screening platform that can be used to analyze interactions between proteins. Yeast display is a versatile platform for high throughput protein engineering and was used to study a broad range of proteins, including antibodies, receptors, enzymes, and model proteins. However, displaying unstable and transient protein complexes on yeast surface is challenging, especially for those protein complexes that weakly associate with each other and has a high dissociation constant. We demonstrated that an engineered intersubunit disulfide bond can be used to covalently crosslink transient and unstable protein complexes displayed on the yeast display. The displayed complex can be verified and quantified by flow cytometry. We also demonstrated that the formation of an intersubunit disulfide bond is highly specific and depends on brings the interacting cysteine residues to close proximity. Together, we showed that disulfide trapping can be used to stabilize transient and unstable protein complexes and the combination with yeast surface and flow cytometry may be useful for studying protein-protein interaction. In the last section of the dissertation, we evaluated specific enzyme-substrate relationship based on measuring the fluorescence resonance energy transfer (FRET) that varies with the level of posttranslational modification (PTM). The use of FRET for the detection of PTM has been demonstrated in previous studies, which showed that the strategy is general and can be used to study a large array of posttranslational modifications on a common platform. However, these methods used in these studies are not applicable to a high throughput analysis of enzyme-substrate relationship because they use low throughput methods, such as fluorescence spectroscopy or microscopy for monitoring the changes in fluorescence. In this study, we have demonstrated that the substrate specificity of a PTM enzyme can be characterized in vitro and in cell using a genetic FRET detector, which consists of a short peptide linked with a phosphotheorine binding protein, sandwiched by two fluorescence proteins that can be detected and quantified by flow cytometry. This study shows that flow cytometry can be used to quantify FRET efficiency to a near single residue resolution and therefore, is useful in identifying the sequences that are preferentially targeted for PTM. Such high throughput assay can be used as a robust molecular tool to characterize the mechanism of substrate recognition during various biologically relevant PTM processes.