Green fluorescent protein (GFP) fusion proteins provide a potentially facile tool for identification of well expressed, properly behaved membrane proteins for biochemical and structural study. Here, we present a GFP-expression survey of >300 membrane proteins from 18 bacterial and archaeal extremophiles, organisms expected to be rich sources of membrane proteins having robust biophysical properties. We find that GFP-fusion fluorescence intensity is an excellent indicator of over-expression potential. By employing a follow-up optimization protocol using a suite of non-GFP constructs and different expression temperatures, we obtain 0.5-15 mg L 21 expression levels for 90% of the tested candidate proteins that pass the GFP screen. Evaluation of the results suggests that certain organisms may serve as better sources of wellexpressed membrane proteins than others, that the degree to which codon usage matches the expression host is uncorrelated with success rate, and that the combination of GFP screening and expression optimization is essential for producing biochemically tractable quantities of material.

Erectile dysfunction adversely affects up to 20% of all men and is the most commonly treated sexual disorder. The public health implications of this condition are significant and represent a challenge for our healthcare system. The physiological pathways responsible for erections have been extensively studied, and much advancement has been made since the introduction of phosphodiesterase 5 inhibitors. Newer agents, such as dopaminergic and melanocortin receptor agonists, which target central erectogenic pathways, are under investigation. Newer formulations and delivery methods of existing medications such as alprostadil will also be introduced in the near future. Furthermore, low-intensity shockwave lithotripsy and stem cell regenerative techniques are innovative approaches to the treatment of erectile dysfunction.

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Biological membranes organize and compartmentalize cell signaling into discrete microdomains, a process that often involves stable, cholesterol-rich platforms that facilitate protein-protein interactions. Polarized cells with distinct apical and basolateral cell processes rely on such compartmentalization to maintain proper function. In the cochlea, a variety of highly polarized sensory and non-sensory cells are responsible for the early stages of sound processing in the ear, yet little is known about the mechanisms that traffic and organize signaling complexes within these cells. We sought to determine the prevalence, localization, and protein composition of cholesterol-rich lipid microdomains in the cochlea. Lipid raft components, including the scaffolding protein caveolin and the ganglioside GM1, were found in sensory, neural, and glial cells. Mass spectrometry of detergent-resistant membrane (DRM) fractions revealed over 600 putative raft proteins associated with subcellular localization, trafficking, and metabolism. Among the DRM constituents were several proteins involved in human forms of deafness including those involved in ion homeostasis, such as the potassium channel KCNQ1, the co-transporter SLC12A2, and gap junction proteins GJA1 and GJB6. The presence of caveolin in the cochlea and the abundance of proteins in cholesterol-rich DRM suggest that lipid microdomains play a significant role in cochlear physiology.