Description
In a previous project, an in vivo chimeric screen for protein stability was developed that has been shown to discriminate both stabilized and destabilized variants of a small wild-type protein: Streptococcal protein G (G_1). In this thesis project, we have replaced G_1 in the chimeric screen with another small test protein referred to as the B domain of native Staphylococcal protein A (SpAB) in order to further test the parameters of the chimeric screen. Another focus of this project was to explore the biophysical properties of these two proteins and to compare and contrast their mechanical and thermal properties as an additional function of the screen. To achieve these goals a number of biophysical and biochemical techniques were applied to measure parameters such as the protein melting temperature, and proteolytic resistance. The SpAB and G_1 protein domains are ideal for this study as they are both small, similar in size, and both have well defined secondary structure, as well as tertiary structure. We have previously generated a set of approximately 20 G_1 mutant variants span a thermal stability range from as low as 28_ C to greater than 100_ C. For this project we have also generated 14 SpAB variants that span a range of thermal stability range of from < 20_ C to 76_ C. Experiments using the SpAB variants will allow us to compare properties of two different proteins (i.e. G_1 and SpAB) that have similar thermal properties but different secondary structures, and mechanical properties. This comparison will ultimately generate valuable data for the field of protein design as it will provide a critical comparison of the differences between a predominantly _-sheet protein and one that is solely _-helical. Within the scope of this thesis project, it was discovered that the catalytic properties of a specific enzyme, human C-terminal hydrolase (UCHL3) hydrolytically cleaves ubiquitin protein chimeras in which the protein ubiquitin is fused to the N-terminus of wild-type SpAB and variants. This hydrolysis can be measured discriminately as a function of time and is based solely on the thermal stability of each C-terminal fused SpAB variant.
