Celebrity Politicians as Health-Promoting Role Models in the Media: the Cases of Vladimir Putin, Donald Trump, and Benjamin Netanyahu.

In recent years, scholars have increasingly revealed the importance of celebrities in society, among them celebrity politicians. These celebrities not only influence political attitudes but also serve as role models for many individuals. Yet, little is known regarding what types of role models' politicians serve as in the context of health.

One-step purification of a functional, constitutively activated form of visual arrestin.

Desensitization of agonist-activated G protein-coupled receptors (GPCRs) requires phosphorylation followed by the binding of arrestin, a ~48 kDa soluble protein. While crystal structures for the inactive, 'basal' state of various arrestins are available, the conformation of 'activated' arrestin adopted upon interaction with activated GPCRs remains unknown. As a first step towards applying high-resolution structural methods to study arrestin conformation and dynamics, we have utilized the subtilisin prodomain/Profinity eXact™ fusion-tag system for the high-level bacterial expression and one-step purification of wild-type visual arrestin (arrestin 1) as well as a mutant form (R175E) of the protein that binds to non-phosphorylated, light-activated rhodopsin (Rho∗).

Designing point mutants to detect structural coupling in a heterotrimeric G protein alpha-subunit by NMR spectroscopy.

To better understand the mechanism by which the activating signal is transmitted from the receptor-interacting regions on the G protein alpha-subunit (G(alpha)) to the guanine nucleotide-binding pocket, we generated and characterized mutant forms of G(alpha) with alterations in switch II (Trp-207-->Phe) and the carboxyl-terminus (Phe-350-->Ala). Previously reported bacterial expression methods for the high-level production of a uniformly isotope-labeled G(talpha)/G(i1alpha) chimera, ChiT, were successfully used to isolate milligram quantities of (15)N-labeled mutant protein. NMR analysis showed that while the GDP/Mg(2+)-bound state of both mutants shared an overall conformation similar to that of the GDP/Mg(2+)-bound state of ChiT, formation of the "transition/activated" state in the presence of aluminum fluoride (AlF(4) (-)) revealed distinct differences between the wild-type and mutant G(alpha) subunits, particularly in the response of the (1)HN, (15)N cross-peak for the Trp-254 indole in the Trp-207-->Phe mutant and the (1)HN, (15)N cross-peak for Ala-350 in the Phe-350-->Ala mutant.

The receptor-bound "empty pocket" state of the heterotrimeric G-protein alpha-subunit is conformationally dynamic.

Heterotrimeric G-protein activation by a G-protein-coupled receptor (GPCR) requires the propagation of structural signals from the receptor-interacting surfaces to the guanine nucleotide-binding pocket. To probe conformational changes in the G-protein alpha-subunit (G(alpha)) associated with activated GPCR (R*) interactions and guanine nucleotide exchange, high-resolution solution NMR methods are being applied in studying signaling of the G-protein, transducin, by light-activated rhodopsin. Using these methods, we recently demonstrated that an isotope-labeled G(alpha) reconstituted heterotrimer forms functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin and a soluble mimic of R*, both of which trigger guanine nucleotide exchange [Ridge, K.

NMR analysis of rhodopsin-transducin interactions.

Heterotrimeric G-protein activation by an agonist-stimulated G-protein coupled receptor (R*) requires the propagation of structural signals from the receptor interacting surfaces to the guanine nucleotide-binding pocket. Employing high-resolution NMR methods, we are probing heterotrimer-associated and rhodopsin-stimulated changes in an isotope-labeled G-protein alpha-subunit (G(alpha)). A key aspect of the work involves the trapping and interrogation of discrete R*-bound conformations of G(alpha).

Conformational changes associated with receptor-stimulated guanine nucleotide exchange in a heterotrimeric G-protein alpha-subunit: NMR analysis of GTPgammaS-bound states.

Solution NMR studies of a (15)N-labeled G-protein alpha-subunit (G(alpha)) chimera ((15)N-ChiT)-reconstituted heterotrimer have shown previously that G-protein betagamma-subunit (G(betagamma)) association induces a "pre-activated" conformation that likely facilitates interaction with the agonist-activated form of a G-protein-coupled receptor (R*) and guanine nucleotide exchange (Abdulaev, N. G., Ngo, T.

Heterotrimeric G-protein alpha-subunit adopts a "preactivated" conformation when associated with betagamma-subunits.

Activation of a heterotrimeric G-protein by an agonist-stimulated G-protein-coupled receptor requires the propagation of structural signals from the receptor binding interface to the guanine nucleotide binding pocket of the G-protein. To probe the molecular basis of this signaling process, we are applying high resolution NMR to track structural changes in an isotope-labeled, full-length G-protein alpha-subunit (G(alpha)) chimera (ChiT) associated with G-protein betagamma-subunit (G(betagamma)) and activated receptor (R(*)) interactions. Here, we show that ChiT can be functionally reconstituted with G(betagamma) as assessed by aluminum fluoride-dependent changes in intrinsic tryptophan fluorescence and light-activated rhodopsin-catalyzed guanine nucleotide exchange.

Bacterial expression and one-step purification of an isotope-labeled heterotrimeric G-protein alpha-subunit.

Heterologous expression systems are often employed to generate sufficient quantities of isotope-labeled proteins for high-resolution NMR studies. Recently, the interaction between the prodomain region of subtilisin and an active, mutant form of the mature enzyme has been exploited to develop a cleavable affinity tag fusion system for one-step generation and purification of full-length soluble proteins obtained by inducible prokaryotic expression. As a first step towards applying high-resolution NMR methods to study heterotrimeric G-protein alpha-subunit (G(alpha)) conformation and dynamics, the utility of this subtilisin prodomain fusion system for expressing and purifying an isotope-labeled G(alpha) chimera (approximately 40 kDa polypeptide) has been tested.

Phototransduction: crystal clear.

Vertebrate visual phototransduction represents one of the best-characterized G-protein-coupled receptor-mediated signaling pathways. Structural analyses of rhodopsin, G protein, arrestin and several other phototransduction components have revealed common folds and motifs that are important for function. Static and dynamic information has been acquired through the application of X-ray diffraction, solution and solid-state nuclear magnetic resonance spectroscopy's, electron and atomic force microscopy's, and a host of indirect structural methods.

Building a stage for interhelical play in rhodopsin.

Biochemical data providing new insights into the packing of helices I and II in the transmembrane domain of rhodopsin reveals the existence of a specific set of size- and charge-sensitive interhelical interactions that influence protein tertiary structure. These findings have broad implications towards understanding the molecular consequences of naturally occurring mutations associated with the retinal degenerative disease autosomal dominant retinitis pigmentosa.

Evidence for structural changes in carboxyl-terminal peptides of transducin alpha-subunit upon binding a soluble mimic of light-activated rhodopsin.

Although a high-resolution crystal structure for the ground state of rhodopsin is now available, portions of the cytoplasmic surface are not well resolved, and the structural basis for the interaction of the cytoplasmic loops with the retinal G-protein transducin (G(t)) is still unknown. Previous efforts aimed at the design, construction, and functional characterization of soluble mimics for the light-activated state of rhodopsin have shown that grafting defined segments from the cytoplasmic region of bovine opsin onto a surface loop in a mutant form of thioredoxin (HPTRX) is sufficient to confer partial G(t) activating potential [Abdulaev et al. (2000) J.

Grafting segments from the extracellular surface of CCR5 onto a bacteriorhodopsin transmembrane scaffold confers HIV-1 coreceptor activity.

Components from the extracellular surface of CCR5 interact with certain macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1) to mediate viral fusion and entry. To mimic these viral interacting site(s), the amino-terminal and extracellular loop segments of CCR5 were linked in tandem to form concatenated polypeptides, or grafted onto a seven-transmembrane bacteriorhodopsin scaffold to generate several chimeras. The chimera studies identified specific regions in CCR5 that confer HIV-1 coreceptor function, structural rearrangements in the transmembrane region that may modulate this activity, and a role for the extracellular surface in folding and assembly.

Functionally discrete mimics of light-activated rhodopsin identified through expression of soluble cytoplasmic domains.

Numerous studies on the seven-helix receptor rhodopsin have implicated the cytoplasmic loops and carboxyl-terminal region in the binding and activation of proteins involved in visual transduction and desensitization. In our continuing studies on rhodopsin folding, assembly, and structure, we have attempted to reconstruct the interacting surface(s) for these proteins by inserting fragments corresponding to the cytoplasmic loops and/or the carboxyl-terminal tail of bovine opsin either singly, or in combination, onto a surface loop in thioredoxin. The purpose of the thioredoxin fusion is to provide a soluble scaffold for the cytoplasmic fragments thereby allowing them sufficient conformational freedom to fold to a structure that mimics the protein-binding sites on light-activated rhodopsin.

[Biochemical characteristics of bovine retina nucleoside diphosphate kinase].

Nucleoside diphosphate kinase (NDP kinase; ATP: NDP phosphotransferase; EC 2.7.4.

Folding and assembly in rhodopsin. Effect of mutations in the sixth transmembrane helix on the conformation of the third cytoplasmic loop.

Previous studies on bovine opsin folding and assembly have identified an amino-terminal fragment, EF(1-232), which folds and inserts into a membrane only after coexpression with its complementary carboxyl-terminal fragment, EF(233-348). To further characterize this interaction, EF(1-232) production was examined upon coexpression with carboxyl-terminal fragments of varying length and/or amino acid composition. These included fragments with incremental deletions of the third cytoplasmic loop (TH(241-348) and EF(249-348)), a fragment composed of the third cytoplasmic loop and sixth transmembrane helix (HF(233-280)), a fragment composed of the sixth and seventh transmembrane helices (FG(249-312)), and EF(233-348) and TH(241-348) fragments with Pro-267 or Trp-265 mutations.

The three-dimensional structures of two isoforms of nucleoside diphosphate kinase from bovine retina.

The crystal structures of two isoforms of nucleoside diphosphate kinase from bovine retina overexpressed in Escherischia coli have been determined to 2.4 A resolution. Both the isoforms, NBR-A and NBR-B, are hexameric and the fold of the monomer is in agreement with NDP-kinase structures from other biological sources.

Light-induced exposure of the cytoplasmic end of transmembrane helix seven in rhodopsin.

A key step in signal transduction in the visual cell is the light-induced conformational change of rhodopsin that triggers the binding and activation of the guanine nucleotide-binding protein. Site-directed mAbs against bovine rhodopsin were produced and used to detect and characterize these conformational changes upon light activation. Among several antibodies that bound exclusively to the light-activated state, an antibody (IgG subclass) with the highest affinity (Ka approximately 6 x 10(-9) M) was further purified and characterized.

Nucleoside diphosphate kinase from bovine retina: purification, subcellular localization, molecular cloning, and three-dimensional structure.

The biochemical and structural properties of bovine retinal nucleoside diphosphate kinase were investigated. The enzyme showed two polypeptides of approximately 17.5 and 18.

[Functional expression of bovine retina adenylate cyclase b fragment cDNA in Escherichia Coli cells].

A 1488-bp fragment of bovine retina guanylate cyclase B gene encoding the catalytic and dimerizing domains as well as part of the protein kinase domain was expressed in Escherichia coli cells. The expression product was obtained as inclusion bodies and solubilized in 6 M guanidine hydrochloride. The fragment of guanylate cyclase B is a dimer close in catalytic activity to the native enzyme.

Functional expression of bovine opsin in the methylotrophic yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris was examined for functional expression of bovine opsin. An expression plasmid was constructed where the bovine opsin gene was placed downstream from the P. pastoris alcohol oxidase 1 gene promoter and fused at its amino-terminus to the acid phosphatase secretion signal.

[Nucleoside diphosphate kinase from bovine retina is a glycoprotein].

Nucleoside diphosphate (NDP) kinase from bovine retina was found to contain carbohydrates. The subunits of NDP kinase were separated by SDS-PAGE, blotted onto an Immobilon-P membrane, and their carbohydrate content was determined. Both subunits contained equal amounts of Gal, Man, Fuc, Gal-NAc, and Glc-NAc.

Examining rhodopsin folding and assembly through expression of polypeptide fragments.

Previous work on the expression of bovine opsin fragments separated in the cytoplasmic region has allowed the identification of specific polypeptide segments that contain sufficient information to fold independently, insert into a membrane, and assemble to form a functional photoreceptor. To further examine the contributions of these and other polypeptide segments to the mechanism of opsin folding and assembly, we have constructed 20 additional opsin gene fragments where the points of separation occur in the intradiscal, transmembrane, and cytoplasmic regions. Nineteen of the fragments were stably expressed in COS-1 cells.