Detection of Protein Interactions at Cellular Membranes Using Total Internal Reflection Ellipsometry

Résumé du livre

The targeting and subsequent interaction of proteins with membranes and membrane bound receptors is central to numerous cellular processes. Membrane bound receptors play a vital function in cell signalling, and are thus a target for many drugs, whereas aberrant membrane interactions play a major role in human amyloid diseases such as Parkinson's disease. The analysis of these interactions in their native environments poses problems, due to the technical difficulty in depositing membranes for analysis by conventional techniques such as, surface plasmon resonance (SPR). The optical method of spectroscopic eNipsometry in its total internal reflection mode (TIRE) is a combination of spectroscopic ellipsometry (SE) and SPR. This offers sensitive detection of binding between a ligand and its receptor, and gives potential solutions to the membrane deposition issues by Langmuir-Schaefer (LS) deposition. Therefore, TIRE enables the study of natively derived membranes in addition to synthetic lipid environments.The aim of this research was to develop the method of TIRE by focusing on the analysis of two distinct types of biological processes: model I tested protein: lipid interactions via amyloid fibrils at natively derived membrane from the neuronal cell line SH-SY5Y; and model II tested protein: protein interactions involved in the targeting and binding of molecular chaperones to cognate receptors, using natively derived chloroplast (CP) membranes. Results of Model I demonstrated how short amyloid fibrils can bring about a toxic gain of function by increased membrane disruption, and Model II showed specific chaperone binding to cognate receptors, providing evidence that there are two predominant chaperone receptors to reside at the outer CP membrane. Together the results demonstrate that TIRE can be used with natively derived membranes to obtain biologically relevant information.The importance of studying membrane proteins is highlighted by the fact that nearly half of the top-selling drugs target membrane proteins, and organelles as potential therapeutic targets. Therefore this research will have a direct impact on the development of new therapeutics and our understanding of disease.