G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.Keywords: G protein-coupled receptor; Escherichia coli; membrane protein; FtsH; genetic engineering; cannabinoid receptor; bradykinin receptor Supplemental material: see www.proteinscience.orgThe array of functions that membrane proteins (MPs) perform in cells is very broad, ranging from the maintenance of cell structure and protection against toxins in the environment to the production of energy and respiration. Consistent with these vital roles, genes for MPs typically account for 15%-30% of an organism's genome. However, <0.5% of the protein structures contained in the Protein Data Bank are of MPs. Furthermore, the great majority of extant MP structures are of prokaryotic proteins, with only about two dozen distinct eukaryotic MP structures currently available to the public (see http:// blanco.biomol.uci.edu/Membrane_Proteins_xtal.html for a well-curated database of known MP structures). The reason for this relative lag in structural biology efforts toward MPs is twofold: The first bottleneck is the determination of suitable crystallization conditions (Loll 2003). The second limitation is the production of sufficient amounts of MP suitable for initiation/optimization of crystallization trials. The latter limitation is particularly pertinent to mammalian MPs, which may be present 6 Present address:

Positively charged trimethylammonium-functionalized mixed monolayer protected clusters (MMPCs) bind DNA through complementary electrostatic interactions, resulting in complete inhibition of DNA transcription of T7 RNA polymerase. DNA was released from the nanoparticle by intracellular concentrations of glutathione, resulting in efficient transcription. The restoration of RNA production was dose-dependent in terms of GSH, with considerable control of the release process possible through variation in monolayer structure. This work presents a new approach to controlled release of DNA, with potential applications in the creation of transfection vectors and gene regulation systems.

A family of nanoparticles featuring surfaces of varying hydrophobicity was synthesized. The efficiency of DNA-binding was determined, demonstrating in a fivefold modulation in binding a 37-mer DNA strand. Nanoparticle-binding causes a reversible conformational change in the DNA structure, as demonstrated by circular dichroism and fluorescence experiments. Furthermore, the affinity of the nanoparticle for the DNA can be regulated by external agents, though stability of the complex is observed at relatively high ionic strengths. Gene regulation as a means of controlling disease states or altering cellular activity has become a realistic goal within medicinal chemistry. Exploration of synthetic molecules capable of DNA-binding has resulted in the development of several classes of designed scaffolds. Notable success in the creation of DNA transcription regulators can be traced to the use of substituted polyamides that are highly sequence-selective (1-5). Alternate approaches in the search for molecules appropriate for interaction with DNA sequences have resulted in the identification of systems capable of covalent modification of the target DNA strand (6,7), as well as peptide and saccharide scaffolds that utilize known DNA-binding components to confer activity to unique sequences (8-13). Significant progress has also been made toward understanding DNA reactivity via the detailed analysis of less functionally diverse systems (14-16), including polyamines (17) and cationic dendrimers (18)(19)(20). Consideration of the structure of the DNA strands, as well as its regulation in vivo; however, suggests one additional structure for DNA-binding has not been sufficiently utilized. Histones are a necessary packaging element for DNA within the nucleus. With their large diameter (approximately 6.5 nm) and correspondingly low surface curvature, they are well suited for interacting with and condensing extensive stretches of DNA (21). However, with the exception of dendrimers, which present synthetic challenges (22), none of the major systems under investigation employs a similar shape. Our research focuses on the creation of nanoparticles as scaffolds for DNA-binding.Mixed monolayer-functionalized gold nanoparticles present a promising structure for the development of DNA-regulating molecules ( Figure 1A) (23), and have already been shown to be highly effective transfection vectors (24). In a previous report, we demonstrated that quaternary ammonium-functionalized nanoparticle binds 37-mer DNA in a non-aggregated, stoichiometric fashion with high affinity (25). Centrifugation of the DNA:nanoparticle complexes provided a molar ratio of 3-4 nanoparticles per 37-mer DNA strand, while dynamic light scattering (DLS) of the bound species confirmed that the complexes were not aggregated, but had a specific and small radius in solution of approximately 10 nm. The affinity of the nanoparticles for the DNA segment was demonstrated by their ability to interrupt transcription by T7 RNA polymerase in vitro. As this polymerase has an affinit...

The mitochondrial tyrosyl-tRNA synthetases (mt TyrRSs) of Pezizomycotina fungi are bifunctional proteins that aminoacylate mitochondrial tRNATyr and are structure-stabilizing splicing cofactors for group I introns. Studies with the Neurospora crassa synthetase (CYT-18 protein) showed that splicing activity is dependent upon Pezizomycotina-specific structural adaptations that form a distinct group I intron-binding site in the N-terminal catalytic domain. Although CYT-18’s C-terminal domain also binds group I introns, it has been intractable to X-ray crystallography in the full-length protein. Here, we determined an NMR structure of the isolated C-terminal domain of the Aspergillus nidulans mt TyrRS, which is closely related to but smaller than CYT-18’s. The structure shows an S4 fold like that of bacterial TyrRSs, but with novel features, including three Pezizomycontia-specific insertions. 15N-1H two-dimensional NMR showed that C-terminal domains of the full-length A. nidulans and GeoBacillus stearothermophilus synthetases do not tumble independently in solution, suggesting restricted orientations. Modeling onto a CYT-18/group I intron co-crystal structure indicates that the C-terminal domains of both subunits of the homodimeric protein bind different ends of the intron RNA, with one C-terminal domain having to undergo a large shift on its flexible linker to bind tRNATyr or the intron RNA on either side of the catalytic domain. The modeling suggests that the C-terminal domain acts together with the N-terminal domain to clamp parts of the intron’s catalytic core, that at least one C-terminal domain insertion functions in group I intron binding, and that some C-terminal domain regions bind both tRNATyr and group I intron RNAs.

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