Action of molecular switches in GPCRs--theoretical and experimental studies.

G protein coupled receptors (GPCRs), also called 7TM receptors, form a huge superfamily of membrane proteins that, upon activation by extracellular agonists, pass the signal to the cell interior. Ligands can bind either to extracellular N-terminus and loops (e.g. glutamate receptors) or to the binding site within transmembrane helices (Rhodopsin-like family). They are all activated by agonists although a spontaneous auto-activation of an empty receptor can also be observed. Biochemical and crystallographic methods together with molecular dynamics simulations and other theoretical techniques provided models of the receptor activation based on the action of so-called "molecular switches" buried in the receptor structure. They are changed by agonists but also by inverse agonists evoking an ensemble of activation states leading toward different activation pathways. Switches discovered so far include the ionic lock switch, the 3-7 lock switch, the tyrosine toggle switch linked with the nPxxy motif in TM7, and the transmission switch. The latter one was proposed instead of the tryptophan rotamer toggle switch because no change of the rotamer was observed in structures of activated receptors. The global toggle switch suggested earlier consisting of a vertical rigid motion of TM6, seems also to be implausible based on the recent crystal structures of GPCRs with agonists. Theoretical and experimental methods (crystallography, NMR, specific spectroscopic methods like FRET/BRET but also single-molecule-force-spectroscopy) are currently used to study the effect of ligands on the receptor structure, location of stable structural segments/domains of GPCRs, and to answer the still open question on how ligands are binding: either via ensemble of conformational receptor states or rather via induced fit mechanisms. On the other hand the structural investigations of homoand heterodimers and higher oligomers revealed the mechanism of allosteric signal transmission and receptor activation that could lead to design highly effective and selective allosteric or ago-allosteric drugs.

YMn2Hx and RMn(2-y)Fe(y)H6 (R = Y, Er) studied by Raman, infrared and inelastic neutron scattering spectroscopies.

YMn2 forms either interstitial YMn2Hx hydrides for x < or = 4.5 or a complex YMn2H6 hydride when submitted to high hydrogen pressure. These compounds have been studied by inelastic neutron scattering (INS) in order to clarify the different modes of H vibration. The INS spectra of YMn2Hx hydrides are strongly dependent on the H content. YMn2H6 and YMn2D6 show broad bands, also observed by Raman and IR spectroscopy, assigned to H-Mn-H (or D) and Mn-H bending and stretching modes. Both ErMn2D6 and ErMn1.8Fe0.2D6 show, in addition to the H vibration mode, an intense band at 215 cm(-1) which has been attributed to a magnetic excitation of Er3+ in view of its momentum transfer dependence.

Formation of hydrides in (Ti(1-x)Zr(x))Co(2.00) (0 < x < 1) pseudobinary alloys.

The exposure of (Ti(1-x)Zr(x))Co(2.00) intermetallic alloys to hydrogen at high pressure caused (Ti(1-x)Zr(x))Co(2.00) (x = 0.50-0.90) hydrides in the alloy. The crystalline structural, electronic, and magnetic properties of parent alloys and of their hydrides were determined by using XRD (X-ray powder diffraction) and XAS (X-ray absorption spectrometry) and by the use of SQUID (a superconducting quantum interference device). Hydrogenation did not alter the crystal structure of the parent alloy, but it did increase the volume of the unit cell. An in situ Co K-edge XAS study of the hydride revealed that the valence state of Co increased during discharge (which is the release of hydrogen from the hydride). Hydrogenation of the parent alloy also reduced the magnetic moment. A possible mechanism of discharge for the hydride is also proposed.

Bio-batteries and bio-fuel cells: leveraging on electronic charge transfer proteins.

Bio-fuel cells are alternative energy devises based on bio-electrocatalysis of natural substrates by enzymes or microorganisms. Here we review bio-fuel cells and bio-batteries based on the recent literature. In general, the bio-fuel cells are classified based on the type of electron transfer; mediated electron transfer and direct electron transfer or electronic charge transfer (ECT). The ECT of the bio-fuel cells is critically reviewed and a variety of possible applications are considered. The technical challenges of the bio-fuel cells, like bioelectrocatalysis, immobilization of bioelectrocatalysts, protein denaturation etc. are highlighted and future research directions are discussed leveraging on the use of electron charge transfer proteins. In addition, the packaging aspects of the bio-fuel cells are also analyzed and the found that relatively little work has been done in the engineering development of bio-fuel cells.

Structural and magnetic properties of DyMn(2)D(6) synthesized under high deuterium pressure.

DyMn(2)D(6) has been prepared by applying high gaseous deuterium pressure on DyMn(2). This phase is isostructural with other RMn(2)D(6) (R = Y, Er) compounds and crystallizes with a K(2)PtCl(6) type structure having an ordered anion and a partially disordered cation arrangement because Dy and half the Mn atoms are randomly substituted in the same 8c site. The reverse susceptibility follows a Curie-Weiss law with an effective moment of 10 µ(B) similar to that of DyMn(2). Short range magnetic order, corresponding to ferromagnetic correlations, is observed in the neutron patterns up to 10 K and can be attributed to Dy-Dy interactions. The decomposition of the deuteride into Mn and DyD(2), studied by thermal gravimetric analysis, occurs between 470 and 650 K. A further deuterium desorption takes place above 920 K.

Early-onset Alzheimer's disease with a de novo mutation in the presenilin 1 gene.

A 32-year-old woman diagnosed with very rapidly progressing early-onset Alzheimer's disease (EOAD), age of onset 29 years, and S170F mutation in presenilin 1 gene (PSEN1) is presented. Neuroimaging conducted 2 years after the first symptoms was typical for the advanced stage of Alzheimer's disease (AD), showing cortical brain atrophy, particularly within hippocampus, frontal and temporal cortex. The unaffected parents of the proband are not carriers of the mutation. The paternity was confirmed by microsatellite typing, strongly suggesting de novo origin of S170F mutation. In silico modeling of S170F mutation impact on presenilin 1 (PS1) transmembrane structure indicates that the mutation considerably alters putative interactions of PS1 with other proteins within gamma-secretase complex.

Crystal structure of rhodopsin: a template for cone visual pigments and other G protein-coupled receptors.

The crystal structure of rhodopsin has provided the first three-dimensional molecular model for a G-protein-coupled receptor (GPCR). Alignment of the molecular model from the crystallographic structure with the helical axes seen in cryo-electron microscopic (cryo-EM) studies provides an opportunity to investigate the properties of the molecule as a function of orientation and location within the membrane. In addition, the structure provides a starting point for modeling and rational experimental approaches of the cone pigments, the GPCRs in cone cells responsible for color vision. Homology models of the cone pigments provide a means of understanding the roles of amino acid sequence differences that shift the absorption maximum of the retinal chromophore in the environments of different opsins.

Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies.

Guanylyl cyclase-activating proteins are EF-hand Ca(2+)-binding proteins that belong to the calmodulin superfamily. They are involved in the regulation of photoreceptor membrane-associated guanylyl cyclases that produce cGMP, a second messenger of vertebrate vision. Here, we investigated changes in GCAP1 structure using mutagenesis, chemical modifications, and spectroscopic methods. Two Cys residues of GCAP1 situated in spatially distinct regions of the N-terminal domain (positions 18 and 29) and two Cys residues located within the C-terminal lobe (positions 106 and 125) were employed to detect conformational changes upon Ca(2+) binding. GCAP1 mutants with only a single Cys residue at each of these positions, modified with N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine, an environmentally sensitive fluorophore, and with (1-oxy-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate, a spin label reagent, were studied using fluorescence and EPR spectroscopy, respectively. Only minor structural changes around Cys(18), Cys(29), Cys(106), and Cys(125) were observed as a function of Ca(2+) concentration. No Ca(2+)-dependent oligomerization of GCAP1 was observed at physiologically relevant Ca(2+) concentrations, in contrast to the observation reported by others for GCAP2. Based on these results and previous studies, we propose a photoreceptor activation model that assumes changes within the flexible central helix upon Ca(2+) dissociation, causing relative reorientation of two structural domains containing a pair of EF-hand motifs and thus switching its partner, guanylyl cyclase, from an inactive (or low activity) to an active conformation.

Molecular dynamics of buspirone analogues interacting with the 5-HT1A and 5-HT2A serotonin receptors.

Three-dimensional (3-D) models of the human serotonin 5-HT1A and 5-HT2A receptors were constructed, energy refined, and used to study the interactions with a series of buspirone analogues. For both receptors, the calculations showed that the main interactions of the ligand imide moieties were with amino acids in transmembrane helix (TMH) 2 and 7, while the main interactions of the ligand aromatic moieties were with amino acids in TMH5, 6 and 7. Differences in binding site architecture in the region of highly conserved serine and tyrosine residues in TMH7 gave slightly different binding modes of the buspirone analogues at the 5-HT1A and 5-HT2A receptors. Molecular dynamics simulations of receptor-ligand interactions indicated that the buspirone analogues did not alter the interhelical hydrogen bonding patterns upon binding to the 5-HT2A receptor, while interhelical hydrogen bonds were broken and others were formed upon ligand binding to the 5-HT1A receptor. The ligand-induced changes in interhelical hydrogen bonding patterns of the 5-HT1A receptor were followed by rigid body movements of TMH2, 4 and 6 relative to each other and to the other TMHs, which may reflect the structural conversion into an active receptor structure.

Mechanism of rhodopsin activation as examined with ring-constrained retinal analogs and the crystal structure of the ground state protein.

The guanine nucleotide-binding protein (G-protein)-coupled receptor superfamily (GPCR) is comprised of a large group of membrane proteins involved in a wide range of physiological signaling processes. The functional switch from a quiescent to an active conformation is at the heart of GPCR action. The GPCR rhodopsin has been studied extensively because of its key role in scotopic vision. The ground state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Light induces cis-trans isomerization and rhodopsin activation. Here we show that rhodopsin regenerated with a ring-constrained 11-cis-retinal analog undergoes photoisomerization; however, it remains marginally active because isomerization occurs without the chromophore-induced conformational change of the opsin moiety. Modeling the locked chromophore analogs in the active site of rhodopsin suggests that the beta-ionone ring rotates but is largely confined within the binding site of the natural 11-cis-retinal chromophore. This constraint is a result of the geometry of the stable 11-cis-locked configuration of the chromophore analogs. These results suggest that the native chromophore cis-trans isomerization is merely a mechanism for repositioning of the beta-ionone ring which ultimately leads to helix movements and determines receptor activation.

alpha-asarone congeners as hypolipidemic agents. Pseudoreceptor versus minireceptor modeling.

The aim of our investigations was to construct activity models of alpha-asarone analogs and related compounds using in treatment of atherosclerosis. The models are based on pseudo- and minireceptor modeling. As a reference set of molecules 20 alpha-asarone analogs were chosen. All of them were flexibly fitted to the four most probable alignments generated from the most active analogs. Subsequent analysis was conducted using pseudoreceptor module Receptor Surface Analysis from Cerius2 package. Statistical analysis based on Genetic Function Approximation revealed several QSAR models. Furthermore, minireceptor program PrGen was applied to the same alignments as above to evaluate atomistic receptor models against virtual particle pseudoreceptor models. Six new compounds with known activities but not included in the QSAR activity model building were used to test the created models.