Bacteriorhodopsin chimeras containing the third cytoplasmic loop of bovine rhodopsin activate transducin for GTP/GDP exchange

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.     

Single-molecule studies of membrane proteins

Characterizing membrane proteins with single-molecule techniques provides structural and functional insights that cannot be obtained with conventional approaches. Recent studies show that atomic force microscopy (AFM) in the context of a 'lab on a tip' enables the measurement of multiple parameters of membrane proteins. This multifunctional tool can be applied to probe the oligomeric states and conformational changes of membrane protein assemblies in their native environment. The ability to determine diverse properties at high spatial resolution facilitates the mapping of structural flexibilities, electrostatic potentials and electric currents. By using the AFM tip as tweezer, it is possible to characterize unfolding and refolding pathways of single proteins and the location of their molecular interactions. These interactions dictate the stability of the protein and might be modulated by ligands that alter the protein's functional state.     

Identification of an indole series of prostaglandin D2 receptor antagonists

A novel indole series of PGD2 receptor (DP receptor) antagonists is presented. Optimization of this series led to the identification of potent and selective DP receptor antagonists. In particular, antagonists 35 and 36 were identified with Ki values of 2.6 and 1.8 nM, respectively. These two antagonists are also potent in a DP functional assay where they inhibit the PGD2 induced cAMP production in platelet rich plasma with IC50 values of 7.9 and 8.6 nM, respectively. The structure-activity relationships of this indole series of DP receptor antagonists will also be discussed.     

Detecting molecular interactions that stabilize native bovine rhodopsin

Using single-molecule force spectroscopy we probed molecular interactions within native bovine rhodopsin and discovered structural segments of well-defined mechanical stability. Highly conserved residues among G protein-coupled receptors were located at the interior of individual structural segments, suggesting a dual role for these segments in rhodopsin. Firstly, structural segments stabilize secondary structure elements of the native protein, and secondly, they position and hold the highly conserved residues at functionally important environments. Two main classes of force curves were observed. One class corresponded to the unfolding of rhodopsin with the highly conserved Cys110-Cys187 disulfide bond remaining intact and the other class corresponded to the unfolding of the entire rhodopsin polypeptide chain. In the absence of the Cys110-Cys187 bond, the nature of certain molecular interactions within folded rhodopsin was altered. These changes highlight the structural importance of this disulfide bond and may form the basis of dysfunctions associated with its absence.     

Co-axial association of recombinant eye lens aquaporin-0 observed in loosely packed 3D crystals

Aquaporin-0 (AQP0) is the major membrane protein in vertebrate eye lenses. It has been proposed that AQP0 tetramers mediate contact between membranes of adjacent lens fiber cells, which would be consistent with the extraordinarily narrow inter-cellular spacing. We have obtained 3D crystals of recombinant bovine AQP0 that diffract to 7.0 A resolution. The crystal packing was determined by molecular replacement and shows that, within the cubic lattice, AQP0 tetramers are associated head-to-head along their 4-fold axes. Oligomeric states larger than the tetramer were also observed in solution by native gel electrophoresis and analytical ultracentrifugation methods. In the crystals, there are no direct contacts between octamers, and it can thus be inferred that crystalline order is mediated solely by the detergent belts surrounding the membrane protein. Across the tetramer-tetramer interface, extracellular loops A and C interdigitate at the center and the perimeter of the octamer, respectively. The octamer structure is compared with that of the recently determined structure of truncated ovine AQP0 derived from electron diffraction of 2D crystals. Intriguingly, also in these crystals, octamers are observed, but with significantly different relative tetramer-tetramer orientations. The interactions observed in the loosely packed 3D crystals reported here may in fact represent an in vivo association mode between AQP0 tetramers from juxtaposed membranes in the eye lens.     

Crystal Structure of Human Collagen XVIII Trimerization Domain: A Novel Collagen Trimerization Fold

Collagens contain a unique triple-helical structure with a repeating sequence -G-X-Y-, where proline and hydroxyproline are major constituents in X and Y positions, respectively. Folding of the collagen triple helix requires trimerization domains. Once trimerized, collagen chains are correctly aligned and the folding of the triple helix proceeds in a zipper-like fashion. Here we report the isolation, characterization, and crystal structure of the trimerization domain of human type XVIII collagen, a member of the multiplexin family. This domain differs from all other known trimerization domains in other collagens and exhibits a high trimerization potential at picomolar concentrations. Strong chain association and high specificity of binding are needed for multiplexins, which are present at very low levels.     

Dynamics of Klebsiella pneumoniae OmpA transmembrane domain: The four extracellular loops display restricted motion behavior in micelles and in lipid bilayers

The transmembrane domain of Klebsiella pneumoniae OmpA (KpOmpA) possesses four long extracellular loops that exhibit substantial sequence variability throughout OmpA homologs in Enterobacteria, in comparison with the highly conserved membrane-embedded β-barrel core. These loops are responsible for the immunological properties of the protein, including cellular and humoral recognition. In addition to key features revealed by structural elucidation of the KpOmpA transmembrane domain in detergent micelles, studies of protein dynamics provide insight into its function and/or mechanism of action. We have investigated the dynamics of KpOmpA in a lipid bilayer, using magic angle spinning solid-state NMR. The dynamics of the β-barrel and loop regions were probed by the spin-lattice relaxation times of the C(α) and C(β) atoms of the serine and threonine residues, and by cross-polarization dynamics. The β-barrel core of the protein is rigid; the C-terminal halves of two of the four extracellular loops (L1 and L3), which are particularly long in KpOmpA, are highly mobile. The other two loops (L2 and L4), which are very similar to their homologs in Escherichia coli OmpA, and the N-terminal halves of L1 and L3 exhibit more restricted motions. We suggest a correlation between the sequence variability and the dynamics of certain loop regions, which accounts for their respective contributions to the structural and immunological properties of the protein.     

Mutation of a raft-targeting signal in the transmembrane region retards transport of influenza virus hemagglutinin through the Golgi

Inclusion of proteins into membrane-rafts favours interactions required for virus assembly but has also been proposed to facilitate vesicular transport of proteins. The hemagglutinin (HA) of influenza virus contains a raft-targeting sequence in the outer leaflet of its transmembrane region. We report that its mutation enhances co-localization of HA with a cis-Golgi marker and retards Golgi-localized processing, such as acquisition of Endo-H resistant carbohydrates and proteolytic cleavage. In contrast, trimerization of the molecule in the ER and transport to the apical membrane were not affected. The second signal for raft-targeting, S-acylation at cytoplasmic cysteines, did not retard HA transport.     

The influence of gender on adults admitted for asthma

Background: In the United States, the prevalence of asthma is not only higher than in most other countries, it also varies greatly between diverse populations. Only limited data exist that examine the variation of outcomes by gender in patients admitted to a hospital for asthma.Objective: This study assessed outcome differences based on gender in adults who were admitted nationally with the primary diagnosis of asthma.Methods: A retrospective cohort study was conducted of all patients who were admitted to a hospital with the primary diagnosis of asthma in 2002-2005 and were reported in the Nationwide Inpatient Sample of the Healthcare Cost and Utilization Project. Patients were excluded if they were aged <18 years or had an additional diagnosis of chronic obstructive pulmonary disease.Results: A total of 590,410 patients (439,991 women, 150,419 men) were included in the study. Patients admitted for asthma were significantly more likely to be female (P < 0.05). Women were significantly older compared with men (mean [SD], 48.5 [17.4] vs 44.6 [17.0] years, respectively), had a longer length of stay (3.44 vs 2.84 days), were more likely to be white (37.9% vs 34.2%), and had a higher total cost of admission ($10,575 vs $9390) (all, P < 0.05). Women were more likely than men to need a tracheostomy (adjusted odds ratio [AOR] = 2.04; 95% CI, 1.77-2.35) and to have a bronchoscopy (AOR =1.12; 95% CI, 1.05-1.21). Men were significantly more likely than women to have arterial blood gases performed (AOR = 1.15; 95% Cl, 1.05-1.27) and to be intubated (AOR = 1.18; 95% Cl, 1.10-1.26) (both, P < 0.05). Men were significantly more likely to be admitted as an emergency admission (AOR = 1.10; 95% Cl, 1.04-1.18) and to die during hospitalization (AOR =1.69; 95% CI, 1.41-2.03).Conclusion: Although they were less likely to be admitted to a hospital, men were more likely to be admitted as an emergency and to experience worse outcomes compared with women, in this study of adults with asthma in the United States.