The biophysical analysis of the oligomerization states of isoprenyl carboxylmethyltransferase, Ste14p, from Saccharomyces cerevisiae and the design and development of biosensor architectures for the characterization of membrane proteins.

摘要

Approximately 30% of the proteome of all organisms is composed of membrane proteins. These membrane proteins serve a variety of functions and are implicated in many diseases. The therapeutic potential of membrane protein targets is so great that >60% of all drug targets are either membrane receptors or ion channels. Thus, the development of high throughput assays and techniques for the screening of compounds against particular membrane protein targets is essential to the cost-effective discovery of new therapeutics. This thesis serves two purposes. The first is to biophysically characterize the oligomerization state of the isoprenylcysteine carboxylmethyltransferase enzyme Saccharomyces cerevisiae, Ste14p. Previous chemical crosslinking, co-immuno precipitation, and co-purification studies indicate Ste14p forms and functions as homodimer or higher order oligomer. In this study, using a His10 and myc3 N-terminally epitope tagged Ste14p protein (His-Ste14p), sedimentation velocity analyses along with size exclusion chromatography coupled with multi-angle light scattering indicated a dynamic system with the most prevalent species being a tetramer. The incorporation of His-Ste14p into phospholipid nanodiscs also indicates sample heterogeneity with nanodiscs containing multiple His-Ste14p proteins per disc. The second purpose of this thesis is to use His-Ste14p as a model protein for the development of biosensor architectures for the high throughput screening of drugs against membrane protein targets. We have successfully demonstrated for the first time that His-Ste14p can be functionally reconstituted into a polyethylene glycol (PEG) supported POPC lipid bilayer. Furthermore, we have developed a highly sensitive particle based flow cytometry assay was developed for the detection of S-adenosyl-L-homocysteine (SAH), a byproduct of the enzymatic reaction of SAM utilizing enzymes. Rigorous optimization yielded a lower detection limit of 100 muM for this approach, which is sufficient for measuring enzymatic activity. Further development of PEG supported lipid bilayers with Ste14p coupled with sensitive methods for assaying activity could lead to the design of a chip for high throughput analysis, not only for Ste14p, but for other membrane protein targets.