The G-protein coupled receptors (GPCRs) are a class of membrane proteins that trigger cellular responses to external stimuli, and are believed to be targets for nearly half of all pharmaceutical drugs on the market. However, little is known regarding their folding and cellular interactions, as well as what factors are crucial for their activity. Further structural characterization of GPCRs has largely been complicated by problems with expression, purification, and preservation of activity in vitro. Previously, we have demonstrated high-level expression (~4 mg/L of culture) of functional human adenosine A 2 a receptor fused to a green fluorescent protein (A 2 aR-GFP) from Saccharomyces cerevisiae. In this work we re-engineered A 2 aR with a purification tag, developed an adequate purification scheme, and performed biophysical characterization on purified receptors. Milligram amounts per liter of culture of A 2 aR and A 2 aR-GFP were functionally expressed in S. cerevisiae, with a C-terminal deca-histidine tag. Lysis procedures were developed for optimal membrane protein solubilization and recovery through monitoring fluorescence of A 2 aR-GFP-His 10 . One-step purification of the protein was achieved through immobilized metal affinity chromatography. After initial solubilization in n-dodecyl-β-D-maltoside (DDM), a combination of added cholesterol hemisuccinate (CHS) in 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS) was required to stabilize the functional state of the protein. Isolated A 2 aR under these conditions was found to be largely alpha-helical, and properly incorporated into a mixed-micelle environment. The A 2 a-His 10 receptor was purified in quantities of 6 +/− 2 mg/L of culture, with ligand-binding yields of 1 mg/L, although all protein bound to xanthine affinity resin. This represents the highest purified total and functional yields for A 2 aR yet achieved from any heterologous expression system.

Infrared spectroscopy has been used in the past to probe the dynamics of internal proton transfer reactions taking place during the functional mechanism of proteins but has remained mostly silent to protonation changes in the aqueous medium. Here, by selectively monitoring vibrational changes of buffer molecules with a temporal resolution of 6 µs, we have traced proton release and uptake events in the light-driven proton-pump bacteriorhodopsin and correlate these to other molecular processes within the protein. We demonstrate that two distinct chemical entities contribute to the temporal evolution and spectral shape of the continuum band, an unusually broad band extending from 2,300 to well below 1,700 cm The first contribution corresponds to deprotonation of the proton release complex (PRC), a complex in the extracellular domain of bacteriorhodopsin where an excess proton is shared by a cluster of internal water molecules and/or ionic E194/E204 carboxylic groups. We assign the second component of the continuum band to the proton uptake complex, a cluster with an excess proton reminiscent to the PRC but located in the cytoplasmic domain and possibly stabilized by D38. Our findings refine the current interpretation of the continuum band and call for a reevaluation of the last proton transfer steps in bacteriorhodopsin.

Human adenosine A(2)a receptor is a member of the G-protein-coupled receptor (GPCR) superfamily of seven-helix transmembrane (TM) proteins. To test general models for membrane-protein folding and to identify specific features of folding and assembly for this representative member of an important and poorly understood class of proteins, we synthesized peptides corresponding to its seven TM domains. We assessed the ability of the peptides to insert into micelles and vesicles and measured secondary structure for each peptide in aqueous and membrane-mimetic environments. CD spectra indicate that each of the seven TM peptides form thermally stable, independent alpha-helical structures in both micelles and vesicles. The helical content of the peptides depends on the nature of the membrane-mimetic environment. Four of the peptides (TM3, TM4, TM5, and TM7) exhibit very high-helical structure, near the predicted maximum for their TM segments. The TM1 peptide also adopts relatively high alpha-helical structures. In contrast, two of peptides, TM2 and TM6, display low alpha helicity. Similarly, the ability of the peptides to insert into membrane-mimetic environments, assayed by intrinsic tryptophan fluorescence and fluorescence quenching, varied markedly. Most peptides exhibit higher alpha helicity in anionic sodium dodecyl sulfate than in neutral dodecyl-beta-D-maltoside micelles, and TM2 was disordered in zwiterionic DMPC but was alpha-helical in negatively charged DMPC/DMPG vesicles. These findings strongly suggest that electrostatic interactions between lipids and peptides control the insertion of the peptides and may be involved in membrane-protein-folding events. The measured helical content of these TM domains does not correlate with the predicted helicity based on amino acid sequence, pointing out that, while hydrophobic interactions can be a major determinant for folding of TM peptides, other factors, such as electrostatic interactions or helix-helix interactions, may play significant roles for specific TM domains. Our results represent a comprehensive analysis of helical propensities for a human GPCR and support models for membrane-protein folding in which interactions between TM domains are required for proper insertion and folding of some TM helix domains. The tendency of some peptides to self-associate, especially in aqueous environments, underscores the need to prevent improper interactions during folding and refolding of membrane proteins in vivo and in vitro.

The human adenosine A 2A receptor (A 2A R) belongs to one of the largest family of membrane proteins, the G-protein coupled receptors (GPCRs), characterized by seven transmembrane (TM) helices. Little is known about the determinants of their structures, folding, assembly, activation mechanisms, and oligomeric states. Previous studies in our group showed that peptides corresponding to all seven TM domains form stable helical structures in detergent micelles and lipid vesicles. However, the peptides behave differently; TM5 is the only peptide to have a ratio [u] 222 /[u] 208 obtained by circular dichroism (CD) spectroscopy >1. This finding suggested to us that TM5 might self-associate. In the present study, we investigate the unique properties of the TM5 domain. We performed detailed analyses of TM5 peptide behavior in membrane-mimetic environments using CD spectroscopy, fluorescence spectroscopy and Fo¨rster resonance energy transfer, and gel electrophoresis. We find that TM5 peptide has the ability to self-associate to form oligomeric structures in various hydrophobic milieus and that these oligomers are highly resistant to temperature and chemical denaturation. We also find that mutation of the full-length A 2A R at position M193, which is located in the fifth TM domain, noticeably alters A 2A R monomer:dimer ratio as observed on SDS-PAGE. Our results suggest that parallel association of TM5 dimers may play a role in the known adenosine A 2A receptor dimerization. This study represents the first evidence of an individual GPCR transmembrane domain self-association.Keywords: membrane proteins; GPCR dimerization; helix association; transmembrane peptide; FRET The human adenosine A 2A receptor (A 2A R) is a member of the G-protein coupled receptor (GPCR) superfamily. GPCRs are integral membrane proteins characterized by seven transmembrane (TM) helices that mediate a plethora of cellular signals across the plasma membrane via coupling to G-proteins. They modulate many physiological processes and are linked to numerous human diseases (Shichida and Imai 1998; Gether 2000; Gurrath 2001), and consequently, are the targets of an increasingly large number of drugs (Gurrath 2001). Until recently, GPCRs were believed to work as monomeric entities, activating G proteins in a 1:1 stoichiometric ratio. However, this classical model of coupling may be oversimplified, since a large body of evidence has shown that many GPCRs exist as homodimers, heterodimers, or even as higher order oligomers (Jones et al. 1998;Jordan and Devi 1999;Bai 2004;Fotiadis et al. 2004). Recent studies have also demonstrated that oligomerization has important effects on GPCRs' functions, including ligand binding, receptor Abbreviations: GPCR, G-protein coupled receptor; TM, transmembrane; A 2A R, adenosine A 2A receptor; SDS, sodium dodecyl sulfate; DMPC, dimyristoyl phosphatidylcholine; CD, circular dichroism; PAGE, Polyacrylamide gel electrophoresis; FRET, Fo¨rster resonance energy transfer; PFO, perfluoro-octanoic acid; T m , temperature of ...

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Bacteriorhodopsin (bR) is a membrane protein found in the archae Halobacterium salinarum. Here, we studied wild type bR and especially the triple mutant bR, 3Glu [E9Q/E194Q/E204Q], in combination with wide gap semiconductor TiO2 for their suitability as efficient light harvester in solar cell. Our differential scanning calorimetry data show thermal robustness of bR wild type and 3Glu mutant, which make them good candidates as photosensitizer in solar cells. Molecular modeling indicates that binding of bR to the exposed oxygen atoms of anatase TiO2 is favorable for electron transfer and directed by local, small distance interactions. A solar cell, based on bR wild type and bR triple mutant immobilized on nanocrystalline TiO2 film was successfully constructed. The photocurrent density-photo voltage (J-V) characteristics of bio-sensitized solar cell (BSSC), based on the wild type bR and 3Glu mutant adsorbed on nanocrystalline TiO2 film electrode were measured. The results show that the 3Glu mutant displays better photoelectric performance compared to the wild type bR, giving a short-circuit photocurrent density (J(sc)) of 0.09 mA/cm2 and the open-circuit photovoltage (V(oc)) 0.35 V, under an illumination intensity of 40 mW/cm2.

The A2A adenosine (A2AR) and D2 dopamine (D2R) receptors form oligomers in the cell membrane and allosteric interactions across the A2AR–D2R heteromer represent a target for development of drugs against central nervous system disorders. However, understanding of the molecular determinants of A2AR–D2R heteromerization and the allosteric antagonistic interactions between the receptor protomers is still limited. In this work, a structural model of the A2AR–D2R heterodimer was generated using a combined experimental and computational approach. Regions involved in the heteromer interface were modeled based on the effects of peptides derived from the transmembrane (TM) helices on A2AR–D2R receptor–receptor interactions in bioluminescence resonance energy transfer (BRET) and proximity ligation assays. Peptides corresponding to TM-IV and TM-V of the A2AR blocked heterodimer interactions and disrupted the allosteric effect of A2AR activation on D2R agonist binding. Protein–protein docking was used to construct a model of the A2AR–D2R heterodimer with a TM-IV/V interface, which was refined using molecular dynamics simulations. Mutations in the predicted interface reduced A2AR–D2R interactions in BRET experiments and altered the allosteric modulation. The heterodimer model provided insights into the structural basis of allosteric modulation and the technique developed to characterize the A2AR–D2R interface can be extended to study the many other G protein-coupled receptors that engage in heteroreceptor complexes.

The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechanism has been studied using the single mutants E9Q, E74Q, E194Q, and E204Q; the triple mutant E9Q/E194Q/E204Q; and the quadruple mutant E9Q/E74Q/E194Q/E204Q. Steady-state difference and deconvoluted Fourier transform infrared spectroscopy has been applied to analyze the M- and N-like intermediates in membrane films maintained at a controlled humidity, at 243 and 277 K at alkaline pH. The mutants E9Q and E74Q gave spectra similar to that of wild type, whereas E194Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q showed at 277 K a N-like intermediate with a single negative peak at 1742 cm(-1), indicating that Asp(85) and Asp(96) are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm(-1) and a negative peak at 1742 cm(-1), revealing the presence of protonated Asp(85) (in an M intermediate environment) and deprotonated Asp(96). These results indicate that in E194Q-containing mutants, the second increase in the Asp(85) pK(a) is inhibited because of lack of deprotonation of the proton release group. Our data suggest that Glu(194) is the group that controls the pK(a) of Asp(85).