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Visual opsins bind 11-cis retinal at an orthosteric site to form rhodopsins but increasing evidence suggests that at least some are capable of binding an additional retinoid(s) at a separate, allosteric site(s). Microspectrophotometric measurements on isolated, dark-adapted, salamander photoreceptors indicated that the truncated retinal analog, β-ionone, partitioned into the membranes of green-sensitive rods; however, in blue-sensitive rod outer segments, there was an enhanced uptake of four or more β-ionones per rhodopsin. X-ray crystallography revealed binding of one β-ionone to bovine green-sensitive rod rhodopsin. Cocrystallization only succeeded with extremely high concentrations of β-ionone and binding did not alter the structure of rhodopsin from the inactive state. Salamander green-sensitive rod rhodopsin is also expected to bind β-ionone at sufficiently high concentrations because the binding site is present on its surface. Therefore, both blue- and green-sensitive rod rhodopsins have at least one allosteric binding site for retinoid, but β-ionone binds to the latter type of rhodopsin with low affinity and low efficacy.

Geological disposal in soft rocks is expected as one of the most practicable methods for isolation of high-level radioactive wastes. Since the country rock around the deep tunnels tends to be heated for a long term due to continuous collapse of nuclides, creep behavior of the soft rocks of impermeable nature under high temperature should be studied. Under different temperatures from 24°C to 95°C, a series of unconfined compression tests was conducted on a tuffaceous rock, Ohya stone, at creep stress ratios ranging from 0.6 to 1.0. The results show that the creep behavior is accelerated by high temperatures over 60°C. That is, for higher temperatures, the time to failure is decreased, while the minimum strain rate is increased. A creep model is then presented, in which attention is paid to the change in strain rate with time. Unique relationships between the minimum strain rate and various creep parameters are used, that appear to be dependent on neither creep stress ratio nor temperature. According to the proposed model, the creep failure will not take place if the creep stress ratio is lower than 0.44.

G-protein-coupled receptors (GPCRs) are involved in a vast variety of cellular signal transduction processes from visual, taste and odor perceptions to sensing the levels of many hormones and neurotransmitters. As a result of agonist-induced conformation changes, GPCRs become activated and catalyze nucleotide exchange within the G proteins, thus detecting and amplifying the signal. GPCRs share a common heptahelical transmembrane structure as well as many conserved key residues and regions. Rhodopsins are prototypical GPCRs that detect photons in retinal photoreceptor cells and trigger a phototransduction cascade that culminates in neuronal signaling. Biophysical and biochemical studies of rhodopsin activation, and the recent crystal structure determination of bovine rhodopsin, have provided new information that enables a more complete mechanism of vertebrate rhodopsin activation to be proposed. In many aspects, rhodopsin might provide a structural and functional template for other members of the GPCR family.

When the two-dimensional crystal of bacteriorhodopsin (bR), purple membrane, is incubated at high temperature (32 degreesC) with a small amount of the neutral detergent octylthioglucoside in the presence of the precipitant ammonium sulfate, a large fraction of the membrane fragments is converted into spherical vesicles with a diameter of 50 nm, which are able to assemble into optically isotropic hexagonal crystals when the precipitant concentration is increased. The vesicularization of purple membrane takes place under such a condition that the miscibility of the detergent to the aqueous phase becomes very low, and we suggest that it is initiated by insertion of the detergent molecules into the membrane. At low temperature, the transformation into the vesicular structure is inhibited and no large crystal is produced directly from membrane/detergent/precipitant mixtures. When a suspension of the spherical vesicles produced at the high temperature is cooled and concentrated below 15 degreesC, however, a birefringent hexagonal crystal is produced that diffracts X-rays beyond 2.5 A resolution. This new crystal belongs to the space group P622 with unit cell dimensions of a=b=104.7 A and c=114.1 A, and it is shown to be made up of stacked planar membranes, in each of which the bR trimers are arranged on a honeycomb lattice and the space among the proteins is filled with the detergent molecules and native lipids. These stacked membranes are suggested to be produced by successive fusion of the spherical vesicles. This implies that the crystallization is achieved without any step for complete solubilization of the protein. The present result offers a unique crystallization method that may be applicable to such membrane proteins that are liable to denature in the presence of an excess amount of detergent.

Recently we discovered that some dark-adapted salamander rods and cones generated an electrical response to the truncated retinal analogue, β-ionone. This finding was tempered by observations that exposure to β-ionone led to pigment bleaching, and that very high concentrations of β-ionone inhibited rod channels in patch experiments. Therefore we examined whether β-ionone could activate the visual pigments by attacking the chromophore-binding pocket or through an interaction at an alternate binding site. Microspectrophotometry showed an accumulation of β-ionone in green-sensitive rod outer segments that increased linearly with bath concentration indicating that β-ionone most likely partitioned into disk membranes. β-Ionone also went into blue-sensitive rod outer segments, however, uptake was higher than in green-sensitive rods at all bath concentrations tested, suggesting that β-ionone bound to at least one site on rhodopsin in addition to partitioning. X-ray diffraction of green-sensitive rod rhodopsin crystallized in the presence of millimolar concentrations of β-ionone revealed a binding site located near the extracellular/intradiskal side of rhodopsin. β-Ionone may have low efficacy and low binding affinity because rhodopsin with ligand bound retained the inactive state conformation. β-Ionone is not a native retinoid, so we also examined the effects of retinol. Dark-adapted green-sensitive rods exposed to retinol lost sensitivity to flashes due to direct rhodopsin activation. In addition, rods exhibited a relative increase in sensitivity to shorter wavelengths, consistent with the ability of retinol to act as an antenna chromophore. Similar effects were seen for a blue-sensitive rod. These results support the presence in opsins of a retinoid-binding site(s) in addition to the chromophore-binding pocket, and suggest that this alternate site(s) mediates a number of distinct ligand-specific effects.