Solid-supported phospholipid bilayers: separation matrix for proteomics applications

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2009-05-15

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This dissertation focuses on the development of biological platforms on which the function and characterization of transmembrane proteins can be performed simultaneously utilizing a biomembrane mimic consisting of a solid supported phospholipid bilayer (SLB). The study centered on the platform development, biophysical measurements of transmembrane proteins and membrane species chromatography. Membrane proteins play an essential role in various cellular and physiological processes. Their normal functions are essential to our health, and many impaired proteins have been related to serious diseases. Gaining a better understanding of membrane proteins is an essential step towards the development of more specific and competent drugs. This research study is divided into two main parts. The first part centered on the creation of a new platform for allowing transmembrane proteins to freely move inside supported lipid bilayers with the same mobility that can be found in vesicle systems. SLBs have been extensively used as model systems to study cell membrane processes because they maintain the same two-dimensional fluidity of lipids within the membrane found in live cells. However, one of the most significant limitations of this platform is its inability to incorporate mobile transmembrane species. Our strategy involves supporting the lipid bilayer on a double cushion, where we not only create a large space to accommodate the transmembrane portion of the protein, but also passivate the underlying substrate to reduce non-physiological protein-substrate interactions. High diffusion constants and high mobile fractions were obtained for a transmembrane protein reconstituted within this double cushion system. The second area of this study focuses on the creation of a new method to rapidly separate membrane components using electrophoresis in SLBs. This work showed that even subtly different chemical isomers can be well-separated by a simple electrophoretic technique when cholesterol is present in the separation matrix. As a first step towards the purification of proteins, this work showed that streptavidin proteins doubly bound to a bilayer by a biotinylated lipid can be separated from streptavidin proteins which are singly bounded.

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