The long-term objective of this project is to design chromatographic methods for the isolation and identification of membrane proteins in order to aid in their proteomic analysis. Presently, membrane proteins are removed from their native biomembrane environment in order to perform electrophoretic separation in gels. This can be unsuitable due to protein aggregation. Indeed, membrane proteins are believed to be underrepresented as a fraction of the proteins that can be typically identified from cells in comparison with their soluble counterparts. To remedy this problem, membrane proteins and associated charged lipids will be separated by electrophoresis in solid supported lipid bilayers. This will maintain these biomacromolecules in a much more native like environment during the entire chromatographic procedure. Lipid bilayers represent a new material for separation and, therefore, it will be necessary to optimize the components of the bilayer which are employed as the separation matrix. Specifically, microdomain structures such as lipid rafts will be exploited in the process because of the differing ability of proteins to partition into liquid expanded and liquid condensed domains. In addition to membrane chemistry, the applied voltage, temperature, buffer conditions, and patterning processes will be tuned. It should also be possible to obtain two-dimensional separation.
The second aim of this work is to develop supports that allow virtually all transmembrane proteins to remain laterally mobile within the planar bilayer environment. Transmembrane proteins, which protrude from the lower leaflet of the supported bilayer, can interact with an underlying inorganic support and become immobile. Two steps will be taken to remedy this problem. First, the bilayer will rest on a well-hydrated polymer cushion that will act to keep membrane proteins in an environment very close to the one found in lipid vesicles. Second, the polymer cushion will be decoupled from the substrate by a passivating protein film.
The third aim of this proposal is to make the separated proteins available for interrogation by ion channel measurements. For this purpose, the isolated proteins bands will be interrogated over a glass nanopore electrode (GNE) approximately 100 to 500 nm in diameter. The final specific aim is to separate proteins from the inner membrane of E. coli. Particular attention will be paid to monitoring transmembrane species from the SEC and Tat translocation apparatuses. We will monitor the ion channel properties of these proteins. It will be necessary to ensure that all membrane proteins are highly vectorially oriented during the separation.
It is generally believed that 15 to 30% of open reading frames in the genomes of most organisms encode membrane proteins. Moreover, 2 out of 3 drug targets are proteins embedded in cellular membranes. It is therefore vital to develop chromatographic assays to identify these species, their expression levels, as well as follow posttranslational modifications.
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|Cho, Younhee; Zhang, Yanjie; Christensen, Trine et al. (2008) Effects of Hofmeister anions on the phase transition temperature of elastin-like polypeptides. J Phys Chem B 112:13765-71|
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