Study of transmembrane protein activity in stabilized lipid membranes and development and applications of surface sensitive plasmon waveguide resonance spectroscopy.

摘要

The work described in this dissertation covers a broad range of research topics all towards realizing the ultimate goal of the establishment of a novel type of biosensor in which the biocompatible membrane structure reconstituted with functional transmembrane proteins is utilized as the sensing element. This thesis focuses on (1) examining the activity of a model transmembrane protein, bovine rhodopsin (Rho) when reconstituted into stabilized lipid membranes in the vesicular geometry and (2) the instrumentation of a novel type of optical spectroscopy, plasmon waveguide resonance (PWR), which is a surface sensitive technique and its application in sensing protein adsorption, reconstitution and activity.;The biophysical properties of lipid membranes have drawn great attention and amount of research is the past few decades due to the crucial roles of membranes in cell function and potential application in biomaterial/biosensor coating. Their properties affect the activity of receptors to a great extent. However, the intrinsic instability of natural or fluid membranes prevents them to be used in a device. Studies have been done to show indirect evidence that the activity of Rho maybe maintained in polymerized membrane composed of bis-SorbPC lipids. The activity of Rho reconstituted into vesicular membranes comprised of various lipids (Egg PC, bis-SorbPC, (poly)bis-SorbPC, mono-SorbPC, (poly)mono-SorbPC, the mixture of Egg PC:bis-SorbPC at 1:1 mol ratio before and after polymerization, the mixture of Egg PC:(poly)bis-SorbPC at 1:1 mol ratio was studied by a more direct technique, UV-Vis. It showed the activity was maintained to 66% of that in natural Egg PC lipid in the mixture of Egg PC:(poly)bis-SorbPC at 1:1 mol ratio as opposed to minimal values in 100 % (poly)lipids.;The new type of spectral PWR was developed based on a commercially available spectrometer with a custom-made flow cell. A silver cladded silica waveguide was utilized as the substrate. The working concept, technical characterization and comparisons with similar techniques are discussed and compared in this work. A modified version of angular PWR in which lipid bilayers are formed by vesicle fusion was also developed. This method excludes any possible effects from a high boiling point organic solvent on either the lipid bilayer itself or the membrane proteins associated with it. A calculating program NphaseAll for spectral PWR was modified based on the surface plasmon resonance program Nphase Fresnel by Corn's group. With this new program, predictions of waveguide properties can be made to provide guidance for waveguide design. Theoretical calculations were done for PWR and experimental results were compared with the theoretical predictions.;PWR was used to detect the formation of a biological lipid membrane, the association of alpha synuclein with membranes and the binding activity of human melanarcortin to its ligands in fluid and polymerized/dried membranes.