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STUDY OF TRANSMEMBRANE PROTEIN ACTIVITY IN STABILIZED LIPID MEMBRANES AND DEVELOPMENT AND APPLICATIONS OF SURFACE SENSITIVE PLASMON WAVEGUIDE RESONANCE SPECTROSCOPYZhang, Han January 2010 (has links)
This dissertation covers a broad range of research topics all towards the ultimate goal establishing of a novel type of biosensor in which the biocompatible membrane structure reconstituted with functional transmembrane proteins is utilized as the sensing element. It focuses on 1) examining the activity of a model transmembrane protein, bovine rhodopsin (Rho) when reconstituted into stabilized lipid membranes 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 biological events.Lipid membrane play crucial roles in cell function. Their biophysical properties affect the activity of a large amount of transmembrane receptors. They are great candidates for biosensing/ biomedical coating. 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 was studied by a more direct technique, UV-Vis. It was found Rho activity was maintained to 66% of that in natural Egg PC lipid in the mixture of Egg PC:(poly)bis-SorbPC (1:1 mol:mol) as opposed to minimal values in 100 % (poly)lipids.A new type of spectral PWR was developed. The working concept, technical characterization and comparisons with similar techniques were discussed and compared in this work. A modified version of angular PWR in which lipid bilayers were formed by vesicle fusion was also developed. This method excludes 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 PWR was developed to do 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.
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Membrane Protein Complexes Involved in Thrombospondin-1 Regulation of Nitric Oxide SignalingGreen, Toni January 2013 (has links)
Thrombospondin-1 (TSP-1) binding to its membrane receptor CD47 results in an inhibtion of the nitric oxide (NO) receptor soluble guanylate cyclase (sGC) and a decrease in intracellular cGMP levels. This causes physiologic effects such as vasoconstriction and a rise in blood pressure. The mechanism by which TSP-1 binds to CD47 at the membrane to decrease sGC activity is largely unknown. CD47 can physically associate with a number of binding partners, including α(v)β₃ and vascular endothelial growth factor receptor 2 (VEGFR2). Binding of a C-terminal fragment of TSP-1 called E3CaG1 to CD47 leads to a rise in intracellular calcium ([Ca²⁺](i)), which decreases sGC activity via a phosphorylation event. Binding of E3CaG1 is also known to disrupt the interaction between CD47 and VEGFR2, leading to a decrease in endothelial nitric-oxide synthase (eNOS) activity and cGMP levels through an Akt signaling pathway. However, it is not known whether other membrane proteins associated with CD47 are required for E3CaG1 binding and a subsequent [Ca²⁺](i) increase. Plasmon-waveguide resonance (PWR) spectroscopy was employed to elucidate the mechanism of TSP-1 inhibition of sGC activity through membrane complexes involving CD47. Using PWR, I found E3CaG1 can bind specifically to CD47 within native Jurkat membranes with picomolar and nanomolar dissociation constants (K(d)), suggesting multiple CD47 complexes are present. Among these complexes, CD47/VEGFR2 was found to bind E3CaG1 with a picomolar K(d)and CD47/α(v)β₃ was found to bind E3CaG1 with a nanomolar K(d). In addition, the presence of an anti-VEGFR2 antibody inhibited the E3CaG1-induced calcium response, which suggested CD47 in complex with VEGFR2 was responsible for TSP-1 reduction of sGC activity. I show that when both CD47 and VEGFR2 are returned to a HEK 293T cell line that does not contain these receptors, an increase in [Ca²⁺](i) upon E3CaG1 binding is restored. Interestingly, E3CaG1 was also found to bind to VEGFR2 in complex with the integrin α(v)β₃ on CD47-null cell lines and their derivations, causing a decrease in [Ca²⁺](i) levels. Therefore, the third type 2 repeat and C-terminal domains of TSP-1 can cause both increases and decreases in calcium based upon the availability of protein complexes to which it binds.
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