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G-protein-coupled receptor domain overexpression in Halobacterium salinarum: Long-range transmembrane interactions in heptahelical membrane proteins
Abstract
The aminergic alpha(2b)-adrenergic receptor (alpha(2b)-AR) third intracellular loop (alpha(2b)-AR 3i) mediates receptor subcellular compartmentalization and signal transduction processes via ligand-dependent interaction with G(i)- and G(o)- proteins. To understand the structural origins of these processes we engineered several lengths of alpha(2b)-AR 3i into the third intracellular loop of the proton pump bacteriorhodopsin (bR) and produced the fusion proteins in quantities suitable for physical studies. The fusion proteins were expressed in the Archaeon Halobacterium salinarum and purified. A highly expressed fusion protein was crystallized from bicelles and diffracted to low resolution on an in-house diffractometer. The bR-alpha(2b)-AR 3i(203-292) protein possessed a photocycle slightly perturbed from that of the wild-type bR. The first half of the fusion protein photocycle, correlated with proton release, is accelerated by a factor of 3, whereas the second half, correlated with proton uptake, is slightly slower than wild-type bR. In addition, there is a large decrease in the pK(a), (from 9.6 to 8.3) of the terminal proton release group in the unphotolyzed state of bR-alpha(2b)-AR 3i as deduced from the pH-dependence of the M-formation. Perturbation of a cytoplasmic loop has thus resulted in the perturbation of proton release at the extracellular surface. The current work indicates that long-range and highly coupled intramolecular interactions exist that are capable of "transducing" structural perturbations (e.g., signals) across the cellular membrane. This gene fusion approach may have general applicability for physical studies of G-protein-coupled receptor domains in the context of the bR structural scaffold.
... Data are presented as the percentage of the GTPg 35 S exchange in the absence of any peptide (the top line). purification, and well characterized structurally (Luecke et al. 1999; Lanyi 2004 ), it is an ideal scaffold for constructing chimeras that contain the elements of rhodopsin (Heymann et al. 2000) or other GPCRs (Jaakola et al. 2005). Multiple lines of evidence suggest that the rhodopsin cytoplasmic loop 3 is an important domain for transducin activation (Franke et al. 1990; Cai et al. 2001; Natochin et al. 2003; Janz and Farrens 2004). ...
... We can therefore readily obtain many milligrams of >95% pure Rh3C by sucrose gradient centrifugation without using a secondary purification method (Fig. 2). Since the purified chimeras are able to maintain the physical properties similar to bR, as determined by UV/Vis and CD spectroscopy (Fig. 3), and a similar migration through sucrose density gradients (Fig. 2B), we suggest that (as also previously suggested by Jaakola et al. 2005) it is possible to overexpress and purify sufficient bR/GPCR chimeras suitable for structural determination. Homotrimers of Rh3C formed in the PM lattice were clearly revealed by the AFM imaging (Fig. 4), indicating that the crystallinity of the lattice was not disrupted by an insertion of the bovine rhodopsin loop 3. The combined data strongly suggest that our bR/Rh chimeras are forming a high-order 2D crystal similar to that of wild-type bR, which may allow for structural determination of the bovine rhodopsin cytoplasmic loop 3 domain. ...
The mechanisms by which G-protein-coupled receptors (GPCRs) activate G-proteins are not well understood due to the lack of atomic structures of GPCRs in an active form or in GPCR/G-protein complexes. For study of GPCR/G-protein interactions, we have generated a series of chimeras by replacing the third cytoplasmic loop of a scaffold protein bacteriorhodopsin (bR) with various lengths of cytoplasmic loop 3 of bovine rhodopsin (Rh), and one such chimera containing loop 3 of the human beta2-adrenergic receptor. The chimeras expressed in the archaeon Halobacterium salinarum formed purple membrane lattices thus facilitating robust protein purification. Retinal was correctly incorporated into the chimeras, as determined by spectrophotometry. A 2D crystal (lattice) was evidenced by circular dichroism analysis, and proper organization of homotrimers formed by the bR/Rh loop 3 chimera Rh3C was clearly illustrated by atomic force microscopy. Most interestingly, Rh3C (and Rh3G to a lesser extent) was functional in activation of GTPgamma35S/GDP exchange of the transducin alpha subunit (Galphat) at a level 3.5-fold higher than the basal exchange. This activation was inhibited by GDP and by a high-affinity peptide analog of the Galphat C terminus, indicating specificity in the exchange reaction. Furthermore, a specific physical interaction between the chimera Rh3C loop 3 and the Galphat C terminus was demonstrated by cocentrifugation of transducin with Rh3C. This Galphat-activating bR/Rh chimera is highly likely to be a useful tool for studying GPCR/G-protein interactions.
It is often necessary to obtain isotopically labeled proteins containing (15)N, (13)C, or (2)H for nuclear magnetic resonance; and (2)H for small-angle neutron scattering or neutron diffraction studies. To achieve uniform isotopic labeling, protein expression is most commonly performed in Escherichia coli or yeast using labeled media. However, proteins from extreme halophiles sometimes require a cellular environment with high ionic strength and cannot be heterologously expressed in E. coli or yeast in functional form. We present here methods for the cultivation of Halobacterium salinarum in isotopically labeled rich media, using commercially available isotopically labeled hydrolysates. The methods described here are both technically simple and relatively inexpensive.
Human Dopamine Receptor D4 (DRD4), a member of G-protein coupled receptor (GPCR) family, plays a central role in cell signaling and trafficking. Dysfunctional activity of DRD4 can lead to several psychiatric conditions and, therefore, represents target for many neurological disorders. However, lack of atomic structure impairs our understanding of the mechanism regulating its activity. Here, we report the modeled structure of DRD4 alone and in complex with dopamine and spiperone, its natural agonist and antagonist, respectively. In order to assess the conformational dynamics induced upon ligand binding, all-atom explicit solvent molecular dynamics simulations in membrane environment were performed. Comprehensive analyses of simulations reveal that agonist binding triggers a series of conformational changes in the transmembrane region, including rearrangement of residues, characteristic of transmission and tyrosine toggle molecular switches. Further, the trajectories indicate that a loop region in the intracellular region - ICL3, is significantly dynamic in nature, mainly due to the side-chain movements of conserved proline residues involved in SH3 binding domains. Interestingly, in dopamine-bound receptor simulation, ICL3 represents an open conformation ideal for G protein binding. The structural and dynamical information presented here suggest a mode of activation of DRD4, upon ligand binding. Our study will help in further understanding of receptor activation, as acquiring structural information is crucial for the design of highly selective DRD4 ligands. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.
The conduct of many trials for the successful production of large quantities of pure proteins for structural biology and biotechnology applications, particularly of active, authentically processed enzymes, large eukaryotic multi-subunit complexes and membrane proteins, has spurred the development of recombinant expression systems. Beyond the well-established Escherichia coli, mammalian cell culture and baculovirus-infected insect cell expression systems, a plethora of alternative expression systems has been discovered, engineered, matured and deployed, resulting in crystal, nuclear magnetic resonance, and electron microscopy structures. In this review, we visit alternative expression hosts for structural biology ranging from bacteria and archaea to filamentous and unicellular yeasts and protozoa, with particular emphasis on their applicability to the structural determination of high-value, challenging proteins and complexes.
The cytoplasmic surface of the G-protein coupled receptor (GPCR) rhodopsin is a key element in membrane receptor activation, molecular recognition by signalling molecules, and receptor deactivation. Understanding of the coupling between conformational changes in the intramembrane domain and the membrane-exposed surface of the photoreceptor rhodopsin is crucial for the elucidation of the molecular mechanism in GPCR activation. As little is known about protein dynamics, particularly the conformational dynamics of the cytoplasmic surface elements on the nanoseconds timescale, we utilised time-resolved fluorescence anisotropy experiments and site-directed fluorescence labelling to provide information on both, conformational space and motion. We summarise our recent advances in understanding rhodopsin dynamics and function using time-resolved fluorescence depolarisation and single molecule fluorescence experiments, with particular focus on the amphipathic helix 8, lying parallel to the cytoplasmic membrane surface and connecting transmembrane helix 7 with the long C-terminal tail.
Adrenergic receptors are integral membrane proteins involved in cellular signalling that belong to the G protein-coupled receptors. Synthetic peptides resembling the putative transmembrane (TM) segments TM4, TM6 and TM7, of the human alpha2-adrenergic receptor subtype C10 (P08913) and defined lipid vesicles were used to assess protein-lipid interactions that might be relevant to receptor structure/function. P6 peptide contains the hydrophobic core of TM6 plus the N-terminal hydrophilic motif REKR, while peptides P4 and P7 contained just the hydrophobic stretches of TM4 and TM7, respectively. All the peptides increase their helical tendency at moderate concentrations of TFE (30-50%) and in presence of 1,2-dielaidoyl-sn-glycero-3-phosphatidylethanolamine (DEPE) lipids. However, only P6 displays up to 19% of alpha-helix in the presence of just the DEPE lipids, evidences a transmembrane orientation and stabilizes the Lalpha lipid phase. Conversely, P4 and P7 peptides form only stable beta-sheet structures in DEPE and favour the non-lamellar, inverted hexagonal (H(II)) phase of DEPE by lowering its phase transition temperature. This study highlights the potential of using synthetic peptides derived from the amino acid sequence in the native proteins as templates to understand the behaviour of the transmembrane segments and underline the importance of interfacial anchoring interactions to meet hydrophobic matching requirements and define membrane organization.
With the advent of the recent determination of high-resolution crystal structures of bovine rhodopsin and human beta2 adrenergic receptor (beta2AR), there are still many structure-function relationships to be learned from other G protein-coupled receptors (GPCRs). Many of the pharmaceutically interesting GPCRs cannot be modeled because of their amino acid sequence divergence from bovine rhodopsin and beta2AR. Structure determination of GPCRs can provide new avenues for engineering drugs with greater potency and higher specificity. Several obstacles need to be overcome before membrane protein structural biology becomes routine: over-expression, solubilization, and purification of milligram quantities of active and stable GPCRs. Coordinated iterative efforts are required to generate any significant GPCR over-expression. To formulate guidelines for GPCR purification efforts, we review published conditions for solubilization and purification using detergents and additives. A discussion of sample preparation of GPCRs in detergent phase, bicelles, nanodiscs, or low-density lipoproteins is presented in the context of potential structural biology applications. In addition, a review of the solubilization and purification of successfully crystallized bovine rhodopsin and beta2AR highlights tools that can be used for other GPCRs.
We have recently shown that the alpha2C10 adrenergic receptor (AR) undergoes short term agonist-promoted desensitization, mediated by phosphorylation of sites in the third intracellular loop. There is significant divergence in the third loop amino acid sequences between alpha2C10 and the other subtypes, alpha2C4 and alpha2C2. We therefore explored the mechanisms of alpha2AR subtype desensitization by expressing each human subtype in Chinese hamster ovary cells and subjecting them to short and long term epinephrine exposures. After 30 min of agonist exposure, alpha2C10 and a2C2 displayed desensitization characterized by rightward shifts in the curves for epinephrine-mediated inhibition of adenylyl cyclase (EC50 = alpha2C10, 0.24 +/- 0.02 muM increasing to 1.1 +/- 0.1 muM; alpha2C2, 1.3 +/- 0.3 increasing to 2.6 +/- 0.3 muM). Coincident with alpha2C10 and alpha2C2 desensitizations were decreases in agonist high affinity binding. In contrast, alpha2C4 underwent no functional desensitization after short term agonist exposure, nor were there any changes in agonist high affinity binding. Agonist-promoted receptor sequestration was clearly greater with alpha2C10 (approximately 26%) and alpha2C2 (approximately 35%) as compared to alpha2C4 (approximately 12%), but such sequestration did not play a significant role in short term desensitization, as blockade with concanavalin A had no effect on desensitization patterns. In contrast to these findings, all alpha2AR subtypes underwent desensitization after prolonged (24 h) agonist exposure. However, alpha2C10 and alpha2C2 displayed substantially more desensitization (approximately 20-fold increase in EC50) as compared to alpha2C4 (approximately 5-fold increase). The primary mechanism of desensitization during long term agonist exposure was found to be a decrease in the amount of cellular G(i), which was equivalent in magnitude in cells expressing all three subtypes. However, in addition to a decrease in G(i), alpha2C10 and alpha2C2 underwent down-regulation of receptor levels during long term agonist exposure, while alpha2C4 did not. Given that all three subtypes bind endogenous catecholamines with high affinity and inhibit adenylyl cyclase efficiently, the significance of multiple subtypes has heretofore been obscure. Our results show that alpha2AR undergo subtype-selective desensitization to agonists and suggest that alpha2AR subtypes may have evolved to meet differing needs for adaptive regulation.
The recently identified natural peptide ligand, tuberoinfundibular peptide of 39 residues (TIP39) for the parathyroid hormone-2 (PTHS) receptor has been structurally characterized by high resolution NMR, circular dichroism, and computer simulations. The structural features of TIP39, determined in the presence of a zwitterionic lipid to mimic the membrane environment of the G-protein-coupled PTH2 receptor, consist of two a-helices, Ala(5)-Arg(21) and Leu(26)-Val(35). Although TIP39 shares limited sequence homology with parathyroid hormone (PTH), a comparison of the structural features of TIP39 and PTH illustrates a similar topological display of residues of the N-terminal helix important for PTH2 receptor activation. The C-terminal helix of TIP39 differs from that of PTH with respect to size and amphipathicity, suggesting an altered mode of binding for TIP39, consistent with the receptor chimera and ligand truncation studies presented in the accompanying paper (Hoare, S. R. J., Clark, J, A, and Usdin, T. B. (2000) J. Biol. Chen. 275, 27274-27283). The structural characterization of TIP39 also provides some insight into the lack of affinity of this novel ligand for the PTH1 receptor.
Previous studies have shown that α2A-adrenergic receptor (α2A-AR) retention at the basolateral surface of polarized MDCKII cells involves its third intracellular (3i loop). The present studies examining mutant α2A-ARs possessing short deletions of the 3i loop indicate that no single region can completely account for the accelerated surface turnover of the Δ3iα2A-AR, suggesting that the entire 3i loop is involved in basolateral retention. Both wild-type and Δ3i loop α2A-ARs are extracted from polarized Madin-Darby canine kidney (MDCK) cells with 0.2% Triton X-100 and with a similar concentration/response profile, suggesting that Triton X-100-resistant interactions of the α2A-AR with cytoskeletal proteins are not involved in receptor retention on the basolateral surface. The indistinguishable basolateral t for either the wild-type or nonsense 3i loop α2A-AR suggests that the stabilizing properties of the α2A-AR 3i loop are not uniquely dependent on a specific sequence of amino acids. The accelerated turnover of Δ3i α2A-AR cannot be attributed to alteration in agonist-elicited α2A-AR redistribution, because α2A-ARs are not down-regulated in response to agonist. Taken together, the present studies show that stabilization of the α2A-AR on the basolateral surface of MDCKII cells involves multiple mechanisms, with the third intracellular loop playing a central role in regulating these processes.
α2-Adrenergic receptors (α2AR) functionally couple not only to Gi but also to Gs. We investigated the amino-terminal portion of the third intracellular loop of the human α2AAR (α2C10) for potential Gs coupling domains using site-directed mutagenesis and recombinant expression in several different cell types. A deletion mutant
and four chimeric receptors consisting of the α2AAR with the analogous sequence from the 5-HT1A receptor (a Gi-coupled receptor) and the β2AR (a Gs-coupled receptor) were expressed in Chinese hamster ovary cells, Chinese hamster fibroblasts, or COS-7 cells and examined
for their ability to mediate stimulation or inhibition of membrane adenylyl cyclase activity or whole cell cAMP accumulation.
In stably expressing Chinese hamster ovary cells, deletion of amino acids 221-231, which are in close proximity to the fifth
transmembrane domain, eliminated α2C10-mediated stimulation of adenylyl cyclase activity, while α2C10-mediated inhibition was only moderately affected. This suggested that this region is important for Gs coupling, prompting construction of the chimeric receptor mutants. Substitution of amino acids 218-235 with 5-HT1A receptor sequence entirely ablated agonist-promoted Gs coupling, as compared with a 338 ± 29% stimulation of adenylyl cyclase activity observed with the wild-type α2C10. In contrast, Gi coupling for this mutant remained fully intact (57 ± 2% versus 52 ± 1% inhibition for wild-type α2C10). Similar substitution with β2AR sequence had no effect on Gi coupling but did reduce Gs coupling. Two additional mutated α2C10 containing smaller substitutions of the amino-terminal region with 5-HT1A receptor sequence at residues 218-228 or 229-235 were then studied. While Gi coupling remained intact with both mutants, Gs coupling was ablated in the former but not the latter mutant receptor. Similar results were obtained using transfected Chinese
hamster fibroblasts (which exclusively display α2AR-Gi coupling) and COS-7 cells (which exclusively display α2AR-Gs coupling). Thus, a critical determinant for Gs coupling is contained within 11 amino acids(218-228) of the amino-terminal region of the third intracellular loop localized
directly adjacent to the fifth transmembrane domain.
Taken together, these studies demonstrate the presence of a discrete structural determinant for agonist-promoted α2AR-Gs coupling, which is distinct and separable from the structural requirements for α2AR-Gi coupling.
The objective is to generate milligram quantities of recombinant human 2C2 adrenergic receptor for X-ray crystallographic studies. It has been cloned in Saccharomyces cerevisiae, and the production level is at best about 13 pmol/mg of membrane protein, as estimated by radio-ligand binding assay. The receptor is solubilized with sucrose monolaurate followed by immunoaffinity purification and reconstitution into phospholipid vesicles. The efficiency of solubilization and immuno-purification are 60% and 91%, respectively.
The sequence of 102 amino acid residues from the NH2 terminus and that of 39 amino acid residues from the COOH terminus of bacteriorhodopsin have been determined. These results are in agreement with those recently published by Ovchinnikov and coworkers [Ovchinnikov, Y.A., Abdulaey, N.G., Feigina, M.Y., Kiselev, A.V. & Lobanov, N.A. (1977) FEBS Lett. 84, 1-4]. Chymotryptic cleavage of bacteriorhodopsin produced two fragments, C-1 (Mr 19,000) and C-2 (Mr 6900), the latter containing the blocked NH2 terminus (pyroglutamic acid). Further fragmentation with CNBr gave mostly hydrophobic fragments, which were separated by gel permeation and reverse-phase high-pressure liquid chromatography in formic acid/ethanol/water mixtures. The fragments were sequenced by a judicious combination of mass spectrometric peptide sequencing and automated Edman degradation. The C-2 fragments were ordered on the basis of methionine-containing peptides identified by gas chromatographic mass spectrometry, while C-1 and C-2 were arranged by analysis of an overlapping CNBr fragment.
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
To investigate the mechanisms of agonist-promoted desensitization of the alpha 2-adrenergic receptor (alpha 2AR), the human alpha 2AAR and a mutated form of the receptor were expressed in CHW cells. After cells were exposed to epinephrine for 30 min, the ability of the wild type alpha 2AAR to mediate inhibition of forskolin-stimulated adenylyl cyclase was depressed by approximately 78%. To assess the role of receptor phosphorylation during desensitization, cells were incubated with 32Pi, exposed to agonist, and alpha 2AAR purified by immunoprecipitation with a fusion protein antibody. Agonist-promoted desensitization was found to be accompanied by phosphorylation of the alpha 2AAR in vivo. The beta-adrenergic receptor kinase (beta ARK) is known to phosphorylate purified alpha 2AAR in vitro. We found that heparin, a beta ARK inhibitor, ablated short term agonist-induced desensitization of alpha 2AAR, while such desensitization was unaffected by inhibition of protein kinase A. To further assess the role of beta ARK, we constructed a mutated alpha 2AAR which has a portion of the third intracellular loop containing 9 serines and threonines (potential phosphorylation sites) deleted. This mutated alpha 2AAR failed to undergo short term agonist-induced desensitization. Agonist promoted in vivo phosphorylation of this mutated receptor was reduced by 90%, consistent with the notion that receptor phosphorylation at sites in the third intracellular loop plays a critical role in alpha 2AAR desensitization. After 24 h of agonist exposure, an even more profound desensitization of alpha 2AAR occurred, which was not accompanied by a decrease in receptor expression. Rather, long term agonist-induced desensitization was found to be due in part to a decrease in the amount of cellular Gi, which was not dependent on receptor third loop phosphorylation sites.
The sedimentation behavior of the halobacterial 7S RNA and bacterioopsin mRNA was assessed after application of total cell lysates to sucrose gradients. These two RNAs cosedimented predominantly with membrane-bound polysomes, and the quantity of 7S RNA bound to the ribosomes was directly correlated with the expression of bacterioopsin. Puromycin treatment released the 7S RNA from the polysomes, indicating that it is transiently associated with protein translation. We suggest that halobacteria contain a signal-recognition-like particle involved in translation of membrane-associated proteins.
The gene coding for bacteriorhodopsin was modified in vitro to replace Asp212 with asparagine and expressed in Halobacterium halobium. X-ray diffraction measurements showed that the major lattice dimension of purple membrane containing the mutated bacteriorhodopsin was the same as wild type. At pH greater than 7, the Asp212----Asn chromophore was blue (absorption maximum at 585 nm) and exhibited a photocycle containing only the intermediates K and L, i.e. a reaction sequence very similar to that of wild-type bacteriorhodopsin at pH less than 3 and the blue form of the Asp85----Glu protein at pH less than 9. Since in the latter cases these effects are attributed to protonation of residue 85, it now appears that removal of the carboxylate of Asp212 has similar consequences as removing the carboxylate of Asp85. However, an important difference is that only Asp85 affects the pKa of the Schiff base. At pH less than 7, the Asp212----Asn protein was purple (absorption maximum at 569 nm) but photoexcitation produced only 15% of the normal amount of M and the transport activity was partial. The reactions of the blue and purple forms after photoexcitation are both quantitatively accounted for by a proposed scheme, K in equilibrium with L1 in equilibrium with L2----BR, but with the addition of an L1 in equilibrium with M reaction with unfavorable pKa for Schiff base deprotonation in the purple form. The latter hinders the transient accumulation of M, and the consequent branching at L1 allows only partial proton transport activity. The results are consistent with the existence of a complex counterion for the Schiff base proposed earlier (De Groot, H. J. M., Harbison, G. S., Herzfeld, J., and Griffin, R. G. (1989) Biochemistry 28, 3346-3353) and suggest that Asp85, Asp212, and at least one other protonable residue participate in it.
Bacteriorhodopsin mutants containing deletions in loop B-C, delta Thr67-Glu74 or delta Gly65-Gln75 or a deletion in the loop E-F, delta Glu161-Ala168, were prepared. Following their expression in Escherichia coli, the mutant proteins were purified to homogeneity and refolded with retinal in detergent-phospholipid mixtures. The mutants containing deletions in the loop B-C were normal at 4 degrees C but showed the following changes at 20 degrees C. 1) The lambda max shifted from 540 to below 510 nm; 2) the rates of bleaching by hydroxylamine in the dark increased; and 3) the rate and steady state of proton pumping decreased. Deletion of the eight amino acids in loop E-F did not affect wild-type behavior. However, all the mutant proteins were more prone to thermal and sodium dodecyl sulfate denaturation than the wild-type bacteriorhodopsin. These observations show that the structures of the B-C and E-F loops are not essential for correct folding of bacteriorhodopsin, but they contribute to the stability of the folded protein.
Above pH 8 the decay of the photocycle intermediate M of bacteriorhodopsin splits into two components: the usual millisecond pH-independent component and an additional slower component with a rate constant proportional to the molar concentration of H+, [H+]. In parallel, the charge translocation signal associated with the reprotonation of the Schiff base develops a similar slow component. These observations are explained by a two-step reprotonation mechanism. An internal donor first reprotonates the Schiff base in the decay of M to N and is then reprotonated from the cytoplasm in the N----O transition. The decay rate of N is proportional to [H+]. By postulating a back reaction from N to M, the M decay splits up into two components, with the slower one having the same pH dependence as the decay of N. Photocycle, photovoltage, and pH-indicator experiments with mutants in which aspartic acid-96 is replaced by asparagine or alanine, which we call D96N and D96A, suggest that Asp-96 is the internal proton donor involved in the re-uptake pathway. In both mutants the stoichiometry of proton pumping is the same as in wild type. However, the M decay is monophasic, with the logarithm of the decay time [log (tau)] linearly dependent on pH, suggesting that the internal donor is absent and that the Schiff base is directly reprotonated from the cytoplasm. Like H+, azide increases the M decay rate in D96N. The rate constant is proportional to the azide concentration and can become greater than 100 times greater than in wild type. Thus, azide functions as a mobile proton donor directly reprotonating the Schiff base in a bimolecular reaction. Both the proton and azide effects, which are absent in wild type, indicate that the internal donor is removed and that the reprotonation pathway is different from wild type in these mutants.
We describe optimization of a transformation system for the halophilic archaebacterium Halobacterium volcanii. Transformation of spheroplasts in the presence of polyethylene glycol permits the uptake and expression of high-molecular-weight linear fragments of genomic DNA as well as plasmid or bacteriophage DNA. Transformations can be performed with either fresh or frozen cell preparations. Auxotrophic mutants were transformed to prototrophy with genomic DNA from wild-type cells with efficiencies of 5 x 10(4)/micrograms of DNA and frequencies of 8 x 10(-5) per regenerated spheroplast. The overall efficiency of transformation with genomic DNA implies that genetic recombination is an efficient process in H. volcanii.
Rhodopsin is a member of an ancient class of receptors that transduce signals through their interaction with guanine nucleotide-binding proteins (G proteins). We have mapped the sites of interaction of rhodopsin with its G protein, which by analogy suggests how other members of this class of receptors may interact with their G proteins. Three regions of rhodopsin's cytoplasmic surface interact with the rod cell G protein transducin (Gt). These are (i) the second cytoplasmic loop, which connects rhodopsin helices III and IV, (ii) the third cytoplasmic loop, which connects rhodopsin helices V and VI, and (iii) a putative fourth cytoplasmic loop formed by amino acids 310-321, as the carboxyl-terminal sequence emerges from helix VII and anchors to the lipid bilayer via palmitoylcysteines 322 and 323. Evidence for these regions of interaction of rhodopsin and Gt comes from the ability of synthetic peptides comprising these regions to compete with metarhodopsin II for binding to Gt. A spectroscopic assay that measures the "extra MII" caused by Gt binding was used to measure the extent of binding of Gt in the presence of competing peptides. The three peptides corresponding to the second, third, and fourth cytoplasmic loops competed effectively with metarhodopsin II, exhibiting Kd values in the 2 microM range; 11 additional peptides comprising all remaining surface regions of rhodopsin failed to compete even at 200 microM. Any two peptides that were effective competitors showed a synergistic effect, having 15 times higher effectiveness when mixed than when assayed separately. A mathematical model was developed to describe this behavior.
The solution structure has been determined for a 19-residue peptide that is fully folded at room temperature. The sequence
of this peptide is based on the C-loop, residues 371-389, of the fourth epidermal growth factor-like domain of thrombomodulin,
a protein that acts as a cofactor for the thrombin activation of protein C. Despite its small size, the peptide forms a compact
structure with almost no repeating secondary structure. The results indicate the structure is held together by hydrophobic
interactions, which in turn stabilize the two β-turns in the structure. The first β-turn in the C-loop represents a conserved
motif that is found in the published structures of five other epidermal growth factor-like proteins. The critical role of
Phe376 in the stabilization of the first β-turn is consistent with mutagenesis data with soluble thrombomodulin. The results also
show that a small subdomain of a larger protein can fold independently, and therefore it could act as an initiation site for
further folding.
The alpha 2A-adrenergic receptor (alpha 2AAR) is polarized to the basolateral membrane of Madin-Darby canine kidney cells via direct targeting. Examination of mutant alpha 2AAR reveals that direct delivery is independent of NH2-terminal glycosylation, COOH-terminal acylation, or protein sequences within the large third cytoplasmic loop or COOH-terminal tail. Combined mutation of these structural features also does not perturb alpha 2AAR delivery, suggesting that a three-dimensional structure imparted by non-contiguous endofacial sequences does not confer alpha 2AAR targeting and that motifs in or near the bilayer must be involved in targeting of the alpha 2AAR. Mutation of a conserved Asp residue in transmembrane two that alters receptor-G-protein interactions also does not impair alpha 2AAR targeting. Finally, modification of sequences in transmembrane seven that resemble tyrosine-containing endocytosis motifs utilized for targeting by some proteins does not perturb alpha 2AAR sorting. Interestingly, deletion of the large third cytoplasmic loop of the alpha 2AAR decreases receptor half-life on the basolateral surface from approximately 11 to 4.5 h without altering the ability of the alpha 2AAR to couple to G-proteins. These data suggest that although targeting of the alpha 2AAR likely involves bilayer sequences, the third cytoplasmic loop may contain structural features that promote stabilization of the alpha 2AAR on the basolateral surface of Madin-Darby canine kidney cells.
Structural changes are central to the mechanism of light-driven proton transport by bacteriorhodopsin, a seven-helix membrane protein. The main intermediate formed upon light absorption is M, which occurs between the proton release and uptake steps of the photocycle. To investigate the structure of the M intermediate, we have carried out electron diffraction studies with two-dimensional crystals of wild-type bacteriorhodopsin and the Asp96-->Gly mutant. The M intermediate was trapped by rapidly freezing the crystals in liquid ethane following illumination with a xenon flash lamp at 5 and 25 degrees C. Here, we present 3.5 A resolution Fourier projection maps of the differences between the M intermediate and the ground state of bacteriorhodopsin. The most prominent structural changes are observed in the vicinity of helices F and G and are localized to the cytoplasmic half of the membrane.
The GPCRDB is a G protein-coupled receptor (GPCR) database system aimed at the collection and dissemination of GPCR related data. It holds sequences, mutant data and ligand binding constants as primary (experimental) data. Computationally derived data such as multiple sequence alignments, three dimensional models, phylogenetic trees and two dimensional visualization tools are added to enhance the database's usefulness. The GPCRDB is an EU sponsored project aimed at building a generic molecular class specific database capable of dealing with highly heterogeneous data. GPCRs were chosen as test molecules because of their enormous importance for medical sciences and due to the availability of so much highly heterogeneous data. The GPCRDB is available via the WWW at http://www.gpcr.org/7tm
Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal bop promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS). The mutagenized bop promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeon Halobacterium sp. strain S9. Active promoters were isolated by screening transformants of an orange (Pum- bop) Halobacterium mutant for purple (Pum+ bop+) colonies on agar plates and analyzed for bop mRNA and/or bacteriorhodopsin content. Sequence analysis yielded the consensus sequence 5'-tyT(T/a)Ta-3', corresponding to the promoter TATA box element 30 to 25 bp 5' of the transcription start site. A putative UAS, 5'-ACCcnactagTTnG-3', located 52 to 39 bp 5' of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement of the TATA box and UAS for bop promoter activity.
The alpha2-adrenergic receptors (alpha2ARs) are localized to and function on the basolateral surface in polarized renal epithelial cells via a mechanism involving the third cytoplasmic loop. To identify proteins that may contribute to this retention, [35S]Met-labeled Gen10 fusion proteins with the 3i loops of the alpha2AAR (Val217-Ala377), alpha2BAR (Lys210-Trp354), and alpha2CAR (Arg248-Val363) were used as ligands in gel overlay assays. A protein doublet of approximately 30 kDa in Madin-Darby canine kidney cells or pig brain cytosol (alpha2B >/= alpha2C> alpha2A) was identified. The interacting protein was purified by sequential DEAE and size exclusion chromatography, and subsequent microsequencing revealed that they are the zeta isoform of 14-3-3 proteins. [35S]Met-14-3-3zeta binds to all three native alpha2AR subtypes, assessed using a solid phase binding assay (alpha2A>/=alpha2B> alpha2C), and this binding depends on the presence of the 3i loops. Attenuation of the alpha2AR-14-3-3 interactions in the presence of a phosphorylated Raf-1 peptide corresponding to its 14-3-3 interacting domain (residues 251-266), but not by its non-phosphorylated counterpart, provides evidence for the functional specificity of these interactions and suggests one potential interface for the alpha2AR and 14-3-3 interactions. These studies represent the first evidence for G protein-coupled receptor interactions with 14-3-3 proteins and may provide a mechanism for receptor localization and/or coordination of signal transduction.
Despite considerable insights concerning the mechanisms regulating short-term agonist-mediated G protein-coupled receptor (GPCR) internalization, little is known about the mechanisms regulating GPCR surface residence over long periods of time. Herein, we experimentally evaluated mechanisms regulating the surface t(1/2) of various alpha(2A)-adrenergic receptor (alpha(2A)AR) structures. The Delta 3i alpha(2A)AR (lacking the third intracellular loop), D79Na(2A)AR (impaired G protein coupling), and CAM alpha(2A)AR (enhanced G protein coupling) all exhibited a cell surface alpha(2A)AR turnover in Chinese hamster ovary cells that was faster than that of the wild type (WT). Cell surface receptor turnover could be slowed with Ligand occupancy of D79N alpha(2A)AR (agonist or antagonist) and CAM alpha(2A)AR (antagonist only) but not the Delta 3i- or WT alpha(2A)AR. This selective ligand-induced surface stabilization was paralleled by a dramatic Ligand-dependent receptor density upregulation for D79N- and CAM alpha(2A)AR structures. Receptors which exhibited surface turnover and density that could be modulated by ligand (D79N and CAM) also demonstrated structural instability, measured by a loss of radioligand binding capacity in detergent solution over time without parallel changes in receptor protein content. In contrast, the shorter surface t(1/2) Of the Delta 3i alpha(2A)AR, whose cell surface t(1/)2 and steady state density were not altered by ligand occupancy, occurred in the context of a structurally stable receptor in detergent solution. These results demonstrate that changes in receptor structure which alter receptor-G protein coupling (either an increase or decrease) are paralleled by structural instability and ligand-induced surface stabilization. These studies also provide criteria for assessing the structural instability of the alpha(2A)AR that can likely be generalized to all GPCRs.
The kinetics of the light-induced proton release in bacteriorhodopsin/lipid/detergent micelles was monitored with the optical pH-indicator fluorescein bound covalently to positions 127-134 (helices D and E and the DE loop) on the extracellular side of the protein (the proton release side). Single cysteine residues were introduced in these positions by site-directed mutagenesis, and fluorescein was attached to the sulfhydryl group by reaction with (iodoacetamido)fluorescein. Two characteristic proton release times (approximately 20 and 70 microseconds) were observed. The faster time constant was recorded when fluorescein was attached to positions 127, 130, 131, 132, and 134, while the slower time was observed with the indicator bound to positions 128, 129, and 133. The results are rationalized by assuming specific helical wheel orientations for helics D and E and by making a choice for the residues in the DE loop: (i) The fast time constants occur with fluorescein either attached to residues 130, 131, and 132 that form the DE loop or when pointing toward the interior of the protein with its aqueous proton channel [residues 127 (helix D) and 134 (helix E)]. (ii) The slower time constants are detected with fluorescein exposed to the exterior lipid/detergent phase when bound to residues 128, 129 (both helix D), and 133 (helix E). This interpretation is supported by measurements of the polarity of the label environment which indicate for fluorescein in group i a more hydrophilic environment and for group ii a more hydrophobic environment. The fastest proton release time (10 microseconds) was observed with fluorescein bound to position 127.(ABSTRACT TRUNCATED AT 250 WORDS)
We investigated the applicability of the green fluorescent protein (GFP) of Aequorea victoria as a reporter for gene expression in an extremely halophilic organism: Halobacterium salinarum. Two recombinant GFPs were fused with bacteriorhodopsin, a typical membrane protein of H. salinarum. These fusion proteins preserved the intrinsic functions of each component, bacteriorhodopsin and GFP, were expressed in H. salinarum under conditions with an extremely high salt concentration, and were proved to be properly localized in its plasma membrane. These results suggest that GFP could be used as a versatile reporter of gene expression in H. salinarum for investigations of various halophilic membrane proteins, such as sensory rhodopsin or phoborhodopsin.
Halobacteria are halophilic representatives of the recently defined domain, the Archaea. Halobacterium salinarium belongs to this group of microorganisms and contains large amounts of bacteriorhodopsin in its membrane. Bacteriorhodopsin is a seven-transmembrane protein that consists of bacterio-opsin (BO), and the chromophore retinal, which is covalently attached to BO. We have investigated whether the expression machinery for BO can be utilized for synthesis of the human β2-adrenoceptor (β2-AR), a protein with a similar seven-transmembrane-helix topology. An expression vector for BO synthesis was modified to express β2-ARs under the control of BO regulatory elements in H.
salinarium. Homologous recombination into the genome was verified by polymerase chain reactions. Northern blots revealed transcripts of the calculated size and significant amounts of epitope-tagged β2-ARs were detected in Western blots. However, binding of the β-AR antagonist 125I-cyanopindolol revealed low levels of functional receptors, and the ligand binding properties of these receptors were altered when compared to native receptors. Expression of chimeras containing larger amino terminal portions of BO did not result in higher receptor levels. Expression of β2-AR in Haloferax volcanii, another member of halobacteria, was achieved with a vector carrying the ferredoxin promoter. The levels of functional receptor as determined by 125I-cyanopindolol binding were »180 fmol/mg protein. The β-AR ligands isoprenaline and propranolol showed affinities expected for functional β2-ARs. Thus, functional human β2-ARs were expressed in halobacteria, constituting a first approach for expression of a eukaryotic protein in the domain of Archaea.
At low bacteriorhodopsin concentration the binding of all-trans-retinal to the apomembrane of Halobacterium halobium, as monitoried by the absorbance change at 568 nm, occurs in a cooperative manner. The simplest way of analyzing the binding data is based on an all-or-none model and results in a Hill coefficient of 3.0 ± 0.2 and an apparent association constant of 2.8 × 106 M-1. The same sigmoidal binding curve was obtained by using the change in circular dichroism at 365 nm (displacement of retinal oxime) or at 263 nm (retinalinduced change in bacterioopsin). Moreover, the trivial explanation of our results, namely, that the sigmoidal shape is caused by a suitably varying extinction coefficient, could be excluded, since the extinction coefficient was shown to be independent of the degree of binding. The Hill coefficient of close to 3 suggests that protein-protein interactions within bacteriorhodopsin trimers are responsible for the observed cooperative effect. Such an interpretation is consistent with the structure of the reconstituted apomembrane which consists of a hexagonal lattice of bacteriorhodopsin in which the bacteriorhodopsin molecules are arranged in clusters of three. The surprisingly small value of the association constant shows that retinal binding to the apomembrane is not irreversible. This was confirmed by exchange experiments between retinal1 and retinal2 which show that bound retinal2 can be displaced by retinal1 and vice versa. At bacteriorhodopsin concentrations much higher than the reciprocal of the association constant, all the retinal added is bound until all the binding sites are occupied. It is therefore possible to determine the extinction coefficient of the chromophore from the slope of the binding curve. The extinction coefficient obtained in this way is based on a knowledge of the retinal concentration and does not depend on a determination of the protein concentration. The resulting value of 62 700 ± 700 M-1 cm-1, at 568 nm, refers to the light-adapted state of the purple membrane at 25 °C in 0.02 M phosphate buffer, pH 6.9, and is corrected for light scattering.
We have recently shown that the alpha 2C10 adrenergic receptor (AR) undergoes short term agonist-promoted desensitization, mediated by phosphorylation of sites in the third intracellular loop. There is significant divergence in the third loop amino acid sequences between alpha 2C10 and the other subtypes, alpha 2C4 and alpha 2C2. We therefore explored the mechanisms of alpha 2AR subtype desensitization by expressing each human subtype in Chinese hamster ovary cells and subjecting them to short and long term epinephrine exposures. After 30 min of agonist exposure, alpha 2C10 and alpha 2C2 displayed desensitization characterized by rightward shifts in the curves for epinephrine-mediated inhibition of adenylyl cyclase (EC50 = alpha 2C10, 0.24 +/- 0.02 microM increasing to 1.1 +/- 0.1 microM; alpha 2C2, 1.3 +/- 0.3 increasing to 2.6 +/- 0.3 microM). Coincident with alpha 2C10 and alpha 2C2 desensitizations were decreases in agonist high affinity binding. In contrast, alpha 2C4 underwent no functional desensitization after short term agonist exposure, nor were there any changes in agonist high affinity binding. Agonist-promoted receptor sequestration was clearly greater with alpha 2C10 (approximately 26%) and alpha 2C2 (approximately 35%) as compared to alpha 2C4 (approximately 12%), but such sequestration did not play a significant role in short term desensitization, as blockade with concanavalin A had no effect on desensitization patterns. In contrast to these findings, all alpha 2AR subtypes underwent desensitization after prolonged (24 h) agonist exposure. However, alpha 2C10 and alpha 2C2 displayed substantially more desensitization (approximately 20-fold increase in EC50) as compared to alpha 2C4 (approximately 5-fold increase). The primary mechanism of desensitization during long term agonist exposure was found to be a decrease in the amount of cellular Gi, which was equivalent in magnitude in cells expressing all three subtypes. However, in addition to a decrease in Gi, alpha 2C10 and alpha 2C2 underwent down-regulation of receptor levels during long term agonist exposure, while alpha 2C4 did not. Given that all three subtypes bind endogenous catecholamines with high affinity and inhibit adenylyl cyclase efficiently, the significance of multiple subtypes has heretofore been obscure. Our results show that alpha 2AR undergo subtype-selective desensitization to agonists and suggest that alpha 2AR subtypes may have evolved to meet differing needs for adaptive regulation.
On the basis of extensive radioligand data and more limited functional data, three pharmacological subtypes of alpha 2-adrenergic receptors have been identified. More recently, three human genes or cDNAs for alpha 2-adrenergic receptors have been identified by molecular cloning. The relationship, however, among the pharmacologically defined subtypes and those identified by molecular cloning has not been clear. In order to resolve this issue, we have compared the pharmacological characteristics of the receptors identified by molecular cloning and expressed in COS-7 cells with the characteristics of the pharmacologically defined receptors in their respective prototypic tissue or cell line. The affinities (Ki values) of 12 subtype-selective alpha 2-adrenergic antagonists were determined for the alpha 2 receptor in the six preparations, by radioligand binding. Correlation analyses of the pKi values indicate that the alpha 2A subtype, as defined in the HT29 cell line, the alpha 2B receptor of the neonatal rat lung, and the alpha 2C subtype, as defined in an oppossum kidney cell line, correspond to the cloned human alpha 2-C10, alpha 2-C2, and alpha 2-C4 receptor subtypes, respectively.
Light-induced H+ release and reuptake as well as surface potential changes inherent in the bacterio-rhodopsin reaction cycle were measured between 10 degrees C and 50 degrees C. Signals of optical pH indicators covalently bound to Lys-129 at the extracellular surface of bacteriorhodopsin were compared with absorbance changes of probes residing in the aqueous bulk phase. Only surface-bound indicators monitor the kinetics of H+ ejection from bacteriorhodopsin and allow the correlation of the photocycle with the pumping cycle. During the L550----M412 transition the H+ appears at the extracellular surface of bacteriorhodopsin. Surface potential changes detected by bound fluorescein or by the potentiometric probe 4-[2-(di-n-butylamino)-6-naphthyl]vinyl-1-(3-sulfopropyl)pyridinium betaine (di-4-ANEPPS) occur in milliseconds concomitantly with the formation and decay of the N intermediate. pH indicators residing in the aqueous bulk phase reflect the transfer of H+ from the membrane surface into the bulk but do not probe the early events of H+ pumping. The observed retardation of H+ at the membrane surface for several hundred microseconds is of relevance for energy conversion of biological membranes powered by electrochemical H+ gradients.
The functional interaction of the recombinant alpha 2 adrenergic receptor subtypes, alpha 2-C10 (the human platelet alpha 2 receptor, equivalent to the alpha 2 A subtype) and alpha 2-C4 (an alpha 2 receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of Gs (adenylyl cyclase stimulatory) and members of the inhibitory family, including Gi1, Gi2, and Gi3, and G0. The cloned alpha subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the alpha subunits with beta gamma subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both alpha 2 receptor subtypes could couple to the inhibitory G proteins but not to Gs, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two receptor subtypes. Between the different inhibitory G proteins, the alpha 2-C10 adrenergic receptor subtype showed the following preference: Gi3 greater than Gi1 greater than or equal to Gi2 greater than G0. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (Gi3) to 1.5-fold (G0). The preference of G-protein interaction for the alpha 2-C4 receptor subtype was the same as that observed for the alpha 2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the alpha 2 adrenergic receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that alpha 2-C10 is coupled more efficiently to G-protein activation than is alpha 2-C4.
Time-resolved difference spectra were measured for Triton X-100 solubilized bacteriorhodopsin monomers between 100 ns and 100 ms after photoexcitation. The results are consistent with the general scheme K in equilibrium L in equilibrium M1 in equilibrium M2 in equilibrium N in equilibrium O----BR proposed previously for purple membranes [Váró, G., & Lanyi, J.K. (1990) Biochemistry 29, 2241-2250]. The rate constants which involve proton release or uptake, i.e., kLM1, kNO, and kON, were significantly higher in the monomeric protein than in purple membrane; the other steps were less affected. Analysis of the temperature dependencies of the rate constants between 5 and 30 degrees C yielded the enthalpies and entropies of activation for all steps except the two absent back-reactions. Comparison of these with data for purple membranes [Váró, G., & Lanyi, J.K. (1991) Biochemistry 30, 5016-5022] shows that the crystalline structure affects the energetics of the photocycle. In bacteriorhodopsin immobilized by the lattice of the purple membrane, the entropy changes leading to all transition states are more positive. Thus, the forward reactions proceed with less conformational hindrance. However, the thermal (enthalpic) barriers are higher. These effects are particularly pronounced for the M1----M2 and O----BR reactions. Large changes of the enthalpy and entropy levels of intermediates in the M2----BR reaction segment, but not in the K----M1 segment, upon solubilization of the protein are consistent with our earlier proposal that major protein conformational changes occur in the photocycle and they begin with the M1----M2 reaction.
The bop gene cluster consists of at least three genes: bop (bacterio-opsin), brp (bacterio-opsin-related protein), and bat (bacterio-opsin activator). We have quantitated transcript levels from these genes in a wild-type and bacterioruberin-deficient mutant of Halobacterium halobium under conditions which affect purple membrane synthesis. In wild-type cultures grown under high oxygen tension in the dark, bop and bat transcript levels were low during steady-state growth and then increased approximately 29- and approximately 45-fold, respectively, upon entry into stationary phase. brp gene transcription remained very low and essentially unchanged under these conditions. In addition, exposure of wild-type cultures growing under high oxygen tension to 30,000 lx of light stimulated expression of all three genes, especially brp. In contrast to the wild-type, transcription from all three genes in the bacterioruberin mutant was very high during steady-state growth under high oxygen tension in the dark. Cultures of the bacterioruberin mutant were shifted at early stationary phase to low oxygen tension to determine whether oxygen concentrations lower than those present in stationary phase would induce transcription of the bop gene cluster in this strain. Indeed, transcription was induced, suggesting that the bop gene cluster is not completely uncoupled from regulation by oxygen tension in the bacterioruberin mutant. From these data, we propose a regulatory model involving two different mechanisms: (i) bat gene expression is induced under conditions of low oxygen tension and the bat gene product activates bop gene expression and (ii) light induces brp transcription, which stimulates or modulates bat transcription.
Homogeneous bacteriorhodopsin was obtained preparatively (100 mg batches) from purple membrane of Halobacterium halobium cells. The homogeneity of the protein was considerably affected by variations in the growth conditions of the bacteria. Fully matured bacteriorhodopsin having a blocked N-terminus and a homogeneous C-terminus, was reproducibly obtained when cells were grown in a sufficiently aerated medium.
On the basis of data obtained by spectroscopic analysis and chromatography of retinal extracts, a consensus has been adopted that dark-adapted purple membrane (pm) contains 13-cis- and all-trans-retinal in equal amounts, whereas the light-adapted membrane contains all-trans-retinal only. We have developed an improved extraction technique which extracts up to 70% of the retinal in pm within 4 min. In the extracts from dark-adapted pm at room temperature, we consistently find 66-67% 13-cis-retinal and 33-34% all-trans-retinal, and more than 98.5% all-trans isomer in light-adapted samples. The spectrum obtained by reconstitution of bacterioopsin with 13-cis-retinal at 2 degrees C (to minimize isomerization) shows an absorbance maximum at 554 nm and agrees well with the spectrum for the 13-cis component calculated from the dark-adapted and light-adapted bR spectra with our extraction data. The ratio of 13-cis:all-trans isomer in dark-adapted pm is 2:1 and nearly constant between 0 and 38 degrees C but begins to decrease distinctly above 40 degrees C, and more rapidly near 70 degrees C, reaching 0.75 at 90 degrees C. The van't Hoff plot of the isomer ratio shows a nonlinear temperature dependence above 40 degrees C, indicating a more complex system than a simple thermal 13-cis/all-trans isomer equilibrium. We attribute the broadening, absorbance decrease, and blut shift of the visible absorption band with increasing temperature to the appearance of at least one and possibly two or three new chromophores which contain, mainly or exclusively, the all-trans isomer.(ABSTRACT TRUNCATED AT 250 WORDS)
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
alpha2-Adrenergic receptors (alpha 2AR) functionally couple not only to Gi but also to Gs. We investigated the amino-terminal portion of the third intracellular loop of the human alpha 2AAR (alpha 2C10) for potential Gs coupling domains using site-directed mutagenesis and recombinant expression in several different cell types. A deletion mutant and four chimeric receptors consisting of the alpha 2AAR with the analogous sequence from the 5-HT1A receptor (a Gi-coupled receptor) and the beta 2AR (a Gs-coupled receptor) were expressed in Chinese hamster ovary cells, Chinese hamster fibroblasts, or COS-7 cells and examined for their ability to mediate stimulation or inhibition of membrane adenylyl cyclase activity or whole cell cAMP accumulation. In stably expressing Chinese hamster ovary cells, deletion of amino acids 221-231, which are in close proximity to the fifth transmembrane domain, eliminated alpha 2C10-mediated stimulation of adenylyl cyclase activity, while alpha 2C10-mediated inhibition was only moderately affected. This suggested that this region is important for Gs coupling, prompting construction of the chimeric receptor mutants. Substitution of amino acids 218-235 with 5-HT1A receptor sequence entirely ablated agonist-promoted Gs coupling, as compared with a 338 +/- 29% stimulation of adenylyl cyclase activity observed with the wild-type alpha 2C10. In contrast, Gi coupling for this mutant remained fully intact (57 +/- 2% versus 52 +/- 1% inhibition for wild-type alpha 2C10). Similar substitution with beta 2AR sequence had no effect on Gi coupling but did reduce Gs coupling. Two additional mutated alpha 2C10 containing smaller substitutions of the amino-terminal region with 5-HT1A receptor sequence at residues 218-228 or 229-235 were then studied. While Gi coupling remained intact with both mutants, Gs coupling was ablated in the former but not the latter mutant receptor. Similar results were obtained using transfected Chinese hamster fibroblasts (which exclusively display alpha 2AR-Gi coupling) and COS-7 cells (which exclusively display alpha 2AR-Gs coupling). Thus, a critical determinant for Gs coupling is contained within 11 amino acids (218-228) of the amino-terminal region of the third intracellular loop localized directly adjacent to the fifth transmembrane domain. Taken together, these studies demonstrate the presence of a discrete structural determinant for agonist-promoted alpha 2AR-Gs coupling, which is distinct and separable from the structural requirements for alpha 2AR-Gi coupling.
The retinal protein halorhodopsin (HR), a light-driven chloride pump from Halobacterium halobium, was homologously overexpressed in this archaebacterium. Two DNA expression systems differing in their promoter region were investigated. The halopsin, hop, promoter coupled to the hop gene gave an increased level of HR synthesis. However, the extent of expression was driven by the copy number of the shuttle vector and did not reach the magnitude of the bacterio-opsin, bop, promoter system. Employing a gene fusion approach, the promoter for the bop gene was used to drive expression of the hop gene. A shuttle vector containing a bop-hop-cartridge was transformed into a HR-deficient strain and blueish-coloured transformants were obtained. The bop promoter expressed HR to an extent where a specific membrane fraction resembled the crystalline purple membrane of BR in terms of the lipid to protein ratio. HR could, therefore, be easily isolated in a natural membrane-bound state. This allows for direct use in biophysical studies without the application of detergents. This was the first successful overexpression of a 7-helical transmembrane protein and may be extended to other proteins of this family.
Because asp-85 is the acceptor of the retinal Schiff base proton during light-driven proton transport by bacteriorhodopsin, modulation of its pKa in the photocycle is to be expected. The complex titration of asp-85 in the unphotolyzed protein was suggested [Balashov, S. P., Govindjee, R., Imasheva, E. S., Misra, S., Ebrey, T. G., Feng, Y., Crouch, R. K., & Menick, D. R (1995) Biochemistry 34, 8820-8834] to reflect the dependence of this residue on the protonation state of another, unidentified group. From the pH dependencies of the rate constant for the thermal equilibration of retinal isomeric states (dark adaptation) and the deprotonation kinetics of the Schiff base during the photocycle in the E204Q and E204D mutants, we identify the residue as glu-204. The nature of its interaction with asp-85 is that at neutral pH either residue can be anionic but not both. This is consistent with our recent finding that glu-204 is the origin of the proton released to the extracellular surface upon protonation of asp-85 during the transport. We propose, therefore, that the following series of events occur in the photocycle. Protonation of asp-85 in the proton equilibrium with the Schiff base of the photoisomerized retinal results in the dissociation of glu-204 and proton release to the extracellular surface. The deprotonation of glu-204, in turn, raises the pK(a) of asp-85, and the equilibrium with the Schiff base shifts toward complete proton transfer. This constitutes the first phase of the reprotonation switch because it excludes asp-85 as a donor in the reprotonation of the Schiff base that follows. The sequential structural changes of the protein that ensue, detected earlier by diffraction, are suggested to facilitate the change of the access of the Schiff base toward the cytoplasmic side as the second phase of the switch, and the lowering the pKa of asp-96, so as to make it a proton donor, as the third phase.
Titration of Asp-85, the proton acceptor and part of the counterion in bacteriorhodopsin, over a wide pH range (2-11) leads us to the following conclusions: 1) Asp-85 has a complex titration curve with two values of pKa; in addition to a main transition with pKa = 2.6 it shows a second inflection point at high pH (pKa = 9.7 in 150-mM KCl). This complex titration behavior of Asp-85 is explained by interaction of Asp-85 with an ionizable residue X'. As follows from the fit of the titration curve of Asp-85, deprotonation of X' increases the proton affinity of Asp-85 by shifting its pKa from 2.6 to 7.5. Conversely, protonation of Asp-85 decreases the pKa of X' by 4.9 units, from 9.7 to 4.8. The interaction between Asp-85 and X' has important implications for the mechanism of proton transfer. In the photocycle after the formation of M intermediate (and protonation of Asp-85) the group X' should release a proton. This deprotonated state of X' would stabilize the protonated state of Asp-85.2) Thermal isomerization of the chromophore (dark adaptation) occurs on transient protonation of Asp-85 and formation of the blue membrane. The latter conclusion is based on the observation that the rate constant of dark adaptation is directly proportional to the fraction of blue membrane (in which Asp-85 is protonated) between pH 2 and 11. The rate constant of isomerization is at least 10(4) times faster in the blue membrane than in the purple membrane. The protonated state of Asp-85 probably is important for the catalysis not only of all-trans <=> 13-cis thermal isomerization during dark adaptation but also of the reisomerization of the chromophore from 13-cis to all-trans configuration during N-->O-->bR transition in the photocycle. This would explain why Asp-85 stays protonated in the N and O intermediates.
Conformational changes are thought to underlie the activation of heterotrimeric GTP-binding protein (G protein)—coupled receptors.
Such changes in rhodopsin were explored by construction of double cysteine mutants, each containing one cysteine at the cytoplasmic
end of helix C and one cysteine at various positions in the cytoplasmic end of helix F. Magnetic dipolar interactions between
spin labels attached to these residues revealed their proximity, and changes in their interaction upon rhodopsin light activation
suggested a rigid body movement of helices relative to one another. Disulfide cross-linking of the helices prevented activation
of transducin, which suggests the importance of this movement for activation of rhodopsin.
Understanding the mechanisms of action of membrane proteins requires the elucidation of their structures to high resolution. The critical step in accomplishing this by x-ray crystallography is the routine availability of well-ordered three-dimensional crystals. We have devised a novel, rational approach to meet this goal using quasisolid lipidic cubic phases. This membrane system, consisting of lipid, water, and protein in appropriate proportions, forms a structured, transparent, and complex three-dimensional lipidic array, which is pervaded by an intercommunicating aqueous channel system. Such matrices provide nucleation sites ("seeding") and support growth by lateral diffusion of protein molecules in the membrane ("feeding"). Bacteriorhodopsin crystals were obtained from bicontinuous cubic phases, but not from micellar systems, implying a critical role of the continuity of the diffusion space (the bilayer) on crystal growth. Hexagonal bacteriorhodopsin crystals diffracted to 3.7 A resolution, with a space group P6(3), and unit cell dimensions of a = b = 62 A, c = 108 A; alpha = beta = 90 degrees and gamma = 120 degrees.
Halobacteria are halophilic representatives of the recently defined domain, the Archaea. Halobacterium salinarium belongs to this group of microorganisms and contains large amounts of bacteriorhodopsin in its membrane. Bacteriorhodopsin is a seven-transmembrane protein that consists of bacterio-opsin (BO), and the chromophore retinal, which is covalently attached to BO. We have investigated whether the expression machinery for BO can be utilized for synthesis of the human beta 2-adrenoceptor (beta 2-AR), a protein with a similar seven-transmembrane-helix topology. An expression vector for BO synthesis was modified to express beta 2-ARs under the control of BO regulatory clements in H. salinarium. Homologous recombination into the genome was verified by polymerase chain reactions. Northern blots revealed transcripts of the calculated size and significant amounts of epitope-tagged beta 2-ARs were detected in Western blots. However, binding of the beta-AR antagonist 125I-cyanopindolol revealed low levels of functional receptors, and the ligand binding properties of these receptors were altered when compared to native receptors. Expression of chimeras containing larger amino terminal portions of BO did not result in higher receptor levels. Expression of beta 2-AR in Haloferax volcanii, another member of halobacteria, was achieved with a vector carrying the ferredoxin promoter. The levels of functional receptor as determined by 125I-cyanopindolol binding were 180 fmol/mg protein. The beta-AR ligands isoprenaline and propranolol showed affinities expected for functional beta 2-ARs. Thus, functional human beta 2-ARs were expressed in halobacteria, constituting a first approach for expression of a eukaryotic protein in the domain of Archaea.
Monoclonal antibodies (Mabs) against human alpha2C2-adrenergic receptor (alpha2C2-AR) were raised in mice and characterized. Bacterially expressed fusion protein consisting a sequence from the putative third intracellular loop (amino acids 213-343) of human alpha2C2 and glutathione-S-transferase (GST) was used as antigen. Results from mass spectrometry of purified thrombin cleaved alpha2C2 polypeptide suggested that the epitope region would lie near the aminoterminal end of the 3rd intracellular loop of human alpha2C2-AR. Elevation of Mabs was detected with Western blotting from mouse blood samples. Three alpha2C2 specific cell clones were expanded to in vitro production in hollow fiber systems. The specificity of the Mabs was further determined by immunoprecipitation and immunocytochemistry. Scatchard analysis of thrombin digested, purified, Europium-labelled antigen (amino acids 213-343 of alpha2C2) revealed binding affinity constants of 0.4 x 10(9), 0.7 x 10(9) and 1.6 x 10(9) M(-1) and Kds of 2.6, 1.4 and 0.6 nM for the three Mabs 2B1, 3G3 and 7G1, respectively.
Lipidic cubic phases provide a continuous three-dimensional bilayer matrix that facilitates nucleation and growth of bacteriorhodopsin microcrystals. The crystals diffract x-rays isotropically to 2.0 angstroms. The structure of this light-driven proton pump was solved at a resolution of 2.5 angstroms by molecular replacement, using previous results from electron crystallographic studies as a model. The earlier structure was generally confirmed, but several differences were found, including loop conformations and side chain residues. Eight water molecules are now identified experimentally in the proton pathway. These findings reveal the constituents of the proton translocation pathway in the ground state.
Rhodopsin is a G protein receptor from a many-membered family of membrane receptors. No high-resolution structure exists for any member of this family due to the insolubility of membrane proteins and the difficulty in crystallizing membrane proteins. Two new approaches to the structure of rhodopsin are described that circumvent these limitations: (1) individual solution structures of the four cytoplasmic domains of rhodopsin are fitted with the transmembrane domain; (2) the solution structure of a complex of the four cytoplasmic domains is determined from nuclear magnetic resonance data. The two structures are similar. To test the validity of these structures, specific site-to-site distances measured on intact membrane-bound rhodopsin are compared to the same distances on the structures reported here. Excellent agreement is obtained. Furthermore, the agreement is obtained with distances measured on the activated form of teh receptor and not with distances on the dark-adapted form of rhodopsin. This approach may prove to have general applicability for the determination of the structure for membrane proteins.
A model for the alpha-carbon positions in the seven transmembrane helices in the rhodopsin family of G-protein-coupled receptors is presented. The model incorporates structural information derived from the analysis of approximately 500 sequences in this family. The location, relative to the centre of the lipid bilayer, of each of the seven helical sequence segments and their probable lengths are deduced from sequence analysis, along with the orientation, relative to the centre of the helix bundle, of each helical segment around its axis. The packing of the helices in the model is guided by the density in a three-dimensional map of frog rhodopsin determined by electron cryo-microscopy. The model suggests which of the residues that are highly conserved in this family of receptors interact with each other. Helices III, V and VI are predicted to protrude more than the others from the central lipid core towards the aqueous phase on the intracellular side of the membrane. This feature could be a property of the receptor structure in some but not all of the conformations that it adopts, since recent studies suggest that relative movement occurs between these helices on photoactivation of rhodopsin. Results from other techniques, including the creation of metal-binding sites and disulphide bridges, site-directed spin-labelling studies, the substituted-cysteine accessibility method and other site-directed mutagenesis studies, are discussed in terms of the model.
Active translocation of ions across membranes requires alternating access of the ion binding site inside the pump to the two membrane surfaces. Proton translocation by bacteriorhodopsin (bR), the light-driven proton pump in Halobacterium salinarium, involves this kind of a change in the accessibility of the centrally located retinal Schiff base. This key event in bR's photocycle ensures that proton release occurs to the extracellular side and proton uptake from the cytoplasmic side. To study the role of protein conformational changes in this reprotonation switch, spin labels were attached to pairs of engineered cysteine residues in the cytoplasmic interhelical loops of bR. Light-induced changes in the distance between a spin label on the EF interhelical loop and a label on either the AB or the CD interhelical loop were observed, and the changes were monitored following photoactivation with time-resolved electron paramagnetic resonance (EPR) spectroscopy. Both distances increase transiently by about 5 A during the photocycle. This opening occurs between proton release and uptake, and may be the conformational switch that changes the accessibility of the retinal Schiff base to the cytoplasmic surface after proton release to the extracellular side.
Alpha 2-adrenergic receptors (alpha 2AARs) are coupled by pertussis-toxin sensitive G proteins to various effectors, including adenylyl cyclase and ion channels. The alpha 2AARs respond to endogenous norepinephrine and epinephrine to elicit a variety of physiological responses, including inhibition of neurotransmitter release, suppression of insulin release from pancreatic beta cells, activation of platelet aggregation, and contraction of arteriolar smooth muscle. Three distinct alpha 2AR subtypes (alpha 2A, alpha 2B, alpha 2C) have been characterized by both pharmacological and molecular biological approaches; however, the lack of subtype-specific ligands has precluded an understanding of the physiological relevance of each subtype. Previous studies demonstrated that mutation of a conserved aspartate residue in the alpha 2AAR to asparagine (D79N alpha 2AAR) resulted in a receptor that retained its ability to inhibit voltage-gated Ca2+ channels and cAMP production but was unable to activate K+ currents in AtT20 cells (Surprenant et al., 1992). To explore the physiological role of the alpha 2AAR subtype and to evaluate the selectivity of alpha 2AAR effects with respect to various signal transduction pathways, we used gene targeting in embryonic stem cells to create a mouse line that expresses the mutant D79N alpha 2AAR instead of the wild-type alpha 2AAR. We established a D79N alpha 2AAR mouse line and characterized various alpha 2AAR-mediated physiological functions in these mutant mice. Because the in vivo D79N alpha 2AAR is expressed at a reduced density relative to wild-type alpha 2A and is not selectively uncoupled from a single signal transduction pathway, our findings of losses of alpha 2AAR-mediated functions in the D79N mice reflect a requirement for the alpha 2AAR subtype but do not reveal the importance of a specific signal transduction pathway. The alpha 2AAR subtype appears to mediate reduction in blood pressure following alpha 2A agonist administration as well as sedative, anesthetic-sparing, and analgesic responses to alpha 2AAR agonists. Therefore, the alpha 2AAR subtype appears to mediate a majority of the clinically relevant responses associated with alpha 2AAR agonist treatment.
We investigated the applicability of the green fluorescent protein (GFP) of Aequorea victoria as a reporter for gene expression in an extremely halophilic organism: Halobacterium salinarum. Two recombinant GFPs were fused with bacteriorhodopsin, a typical membrane protein of H. salinarum. These fusion proteins preserved the intrinsic functions of each component, bacteriorhodopsin and GFP, were expressed in H. salinarum under conditions with an extremely high salt concentration, and were proved to be properly localized in its plasma membrane. These results suggest that GFP could be used as a versatile reporter of gene expression in H. salinarum for investigations of various halophilic membrane proteins, such as sensory rhodopsin or phoborhodopsin.