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Proteomics Just Served Up: Membrane Topology on a Proteome Scale

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If proteomics could be thought of as a smorgasbord, then the membrane proteome is its fermented Baltic herring — tough to deal with initially, but valuable once you’ve acquired the skill to appreciate it. Membrane proteins are themselves consistently fickle and hard to characterize, yet prized as some of the most therapeutically interesting proteins because of their tendency to serve as gatekeepers to the cytoplasm. So it’s no small feat that Gunnar von Heijne and his group at Stockholm University have managed to systematically characterize the orientations of the full complement of membrane proteins in Escherichia coli, the biologist’s favorite bacterium.

In the past, topological analysis of membrane proteins — essentially the study of which parts of a protein are in or outside the cytoplasm, and which parts span the lipid bilayer of the membrane itself — involved sequence-based predictions or tedious experiments to tease out how the protein situated itself in the membrane. But von Heijne and his colleagues in the department of biochemistry and biophysics, and the Stockholm Bioinformatics Center, devised an approach to constrain a sequence-based topology prediction model with data obtained from a series of relatively simple biochemical experiments. For the first time, von Heijne’s team managed to employ the technique to characterize the topology of an organism’s entire membrane proteome.

Von Heijne’s experimental trick involves two types of reporter proteins, alkaline phosphatase and green fluorescent protein, that the members of his group systematically fused to the C-terminus of membrane proteins in E. coli and expressed them in culture. By nature of the reporter proteins’ properties — GFP fluoresces under UV light only when in the cytoplasm, PhoA turns a specific growth medium yellow only when in the periplasm — von Heijne’s team could quite simply record whether the C-terminus of each membrane protein lay in or out of the cytoplasm. When coupled with their in silico predictive model, the results were quite accurate, von Heijne says. “It’s a very easy assay, which allows us to do this in high throughput,” he says.

While the approach works best in E. coli, von Heijne says it should be possible to clone out and express proteins derived from other bacteria in an E. coli system and perform a similar topological analysis. And using a different type of reporter protein, he says, the same type of analysis should also work well in the yeast Saccharomyces cerevisiae.

— John S. MacNeil

Science magazine abstract for "Global Topology Analysis of the Escherichia coli Inner Membrane Proteome"
http://www.sciencemag.org/cgi/content/abstract/308/5726/1321

 

Gunnar von Heijne lab website
http://www.sbc.su.se/gunnar/

 

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