Toshiaki Hamanaka

Design of small-angle X-ray diffractometer using synchrotron radiation at the photon factory

Abstract A demagnifying focusing mirror-monochromator optics has been designed and constructed for time-resolved X-ray diffraction studies of biological substances. Focusing in the vertical plane is performed by a sequence of seven 20 cm long bent mirrors. Focusing in the horizontal plane is performed by a bent triangular silicon crystal monochromator which is placed downstream of the mirror. Phase space calculations predict that the optics should produce a focus 1.0 mm (V)×2.6 mm (H) (4σ-value) at the focus point. When the PF storage ring operates at 2.5 GeV and 0.5 A, the intensity is expected to be 10 11 –10 12 photons/s. A small-angle resolution of better than 1000 A should be obtainable at 1.5 A wavelength with an angular resolution of 0.8 mrad and 0.6 mrad in the vertical and horizontal directions, respectively.

Phase transitions of the purple membrane and the brown holo-membrane X-ray diffraction, circular dichroism spectrum and absorption spectrum studies

Abstract The phase transition of the purple membrane observed by differential scanning calorimetry (Jackson, M.B. and Sturtevant, J.M. (1978) Biochemistry 17, 911–915) has been investigated by X-ray diffraction, circular dichroism and absorption spectrum, in comparison with the phase transition in the brown holo-membrane. The two-dimensional crystal of bacteriorhodopsin transformed into two-dimensional liquid around 74–78°C in the purple membrane and around 50–60°C in the brown holo-membrane. The X-ray diffraction patterns obtained at 78°C for the purple membrane and at 60°C for the brown holo-membrane exhibit several broad peaks. Analysis of the pattern suggests that bacteriorhodopsin molecules aggregate in trimers even above the phase transition temperature. The negative circular dichroism band in the visible region is still present at 80°C in the purple membrane and at 60°C in the brown holo-membrane, but becomes negligibly small at 70°C in the brown holo-membrane. The 560 nm absorption peak due to bacteriorhodopsin changes its position and height drastically around 80°C in the brown holo-membrane as in the purple membrane. X-ray diffraction studies have been made on membranes of total lipids extracted from the purple membrane. No indication of the phase transition has been found between −81°C and 77°C.

On the three visual pigments in the retina of the firefly squid, Watasenia scintillans

SummaryThe deep-sea bioluminescent squid, Watasenia scintillans, has three visual pigments: The major one (A1 pigment) is based on retinal and has λmax = 484 nm, the second one (A2 pigment) is based on 3-dehydroretinal and has λmax = 500 nm, and the third one (A4 pigment) is based on 4-hydroxyretinal and has λmax = 470 nm. The distribution of these 3 visual pigments in the retina was studied by HPLC analysis of the retinals in retina slices obtained by microdissection. It was found that A1 pigment was not located in the specific region of the ventral retina receiving the down-welling light which contains very long photoreceptor cells, forming two strata. A2 and A4 pigment were found exclusively in the proximal pinkish stratum and in the distal yellowish stratum. The role of these pigments in the retina is hypothesized to involve spectral discrimination. The extraction and analysis of retinoids to determine the origin of 3-dehydroretinal and 4-hydroxyretinal in the mature squid showed only a trace amount of 4-hydroxyretinol in the eggs. Similar analysis of other cephalopods collected near Japan showed the absence of A2 or A4 pigment in their eyes.

Volatile anesthetics cause conformational changes of bacteriorhodopsin in purple membrane

We examined the effects of volatile anesthetics on the structure of the bacteriorhodopsin in the purple membrane by measurements of the absorption spectrum and the visible circular dichroism (CD) spectrum and assay of the retinal composition. As the concentrations of halothane, enflurane and methoxyflurane were increased, the absorption at 560 nm decreased but that at 480 nm increased with an isosbestic point around 510 nm. These anesthetic-induced spectroscopic changes were reversible. The CD spectrum showed the biphasic pattern with a positive and a negative band. As the concentration of halothane was increased from 4 mM to 8mM, the negative band reversibly diminished more drastically than the positive band, and at 8 mM of halothane the positive band shifted to around 480 nm. These results show that halothane disturbed the exciton coupling among bacteriorhodopsin molecules. The retinal isomer composition was analyzed using high performance liquid chromatography. The ratio of 13-cis- to all-trans-retinal was 47:53, 34:66 and 19:81 at control, 7.4 mM and 14.9 mM enflurane, respectively. After elimination of enflurane, the ratio returned to the control value. These findings indicate that volatile anesthetic directly affect a bacteriorhodopsin in the purple membrane and induce conformational changes in it.

Time-resolved x-ray diffraction studies on the intensity changes of the 5.9 and 5.1 nm actin layer lines from frog skeletal muscle during an isometric tetanus using synchrotron radiation

Time-resolved x-ray diffraction studies have been made on the 5.9- and 5.1-nm actin layer lines from frog skeletal muscles during an isometric tetanus at 6 degrees C, using synchrotron radiation. The integrated intensities of these actin layer lines were found to increase during a tetanus by 30-50% for the 5.9-nm reflection and approximately 70% for the 5.1-nm reflection of the resting values. The intensity increase of both reflections was greater than that taking place in the transition from rest to rigor state. The intensity change of the 5.9-nm reflection preceded those of the myosin 42.9-nm off-meridional reflection and of the equatorial reflections, as well as the isometric tension development. The intensity profile of the 5.9-nm layer line during contraction was found to be different from that observed in the rigor state.

Fractionation of rhodopsin and other components in the rod outer segment membrane by ammonium sulfate salting-out.

Purified bovine rod outer segment membrane was solubilized in a mixture of 1.5% cholic acid/20% saturated ammonium sulfate and 0.05 M phosphate buffer (pH 7.5). The solubilized rod outer segment membrane was fractionated with ammonium sulfate and 70--90% of rhodopsin (A278/A498= 1.6--1.9) was recovered in the fraction of 50 to 60% saturation with ammonium sulfate, giving a highly concentrated solution of purified rhodopsin (A1CM 498 = 83). By the method of ammonium sulfate salting-out, the solubilized rod outer segment membrane was divided into several fractions without a loss of components. The components in each fraction were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Rhodopsin and opsin amounted to 93% of the total protein in the membrane. Other proteins with molecular weights of 46 000, 52 000, 56 000, 70 000, 95 000, 105 000, 130 000 and 270 000 were also detected. Most of phospholipids in the rod outer segment membrane remained in the supernatant above 60% saturation with ammonium sulfate.

Formation of the two-dimensional hexagonal lattice of bacteriorhodopsin in reconstituted brown membrane

The aggregation state of reconstituted bacteriorhodopsin molecules in the brown membrane has been investigated by X-ray diffraction and CD spectra. It has been confirmed that reconstituted bacteriorhodopsin molecules form the hexagonal lattice spontaneously whereas bacterioopsin molecules do not.

X-ray structural studies of some nonionic detergent micelles

Abstract X-Ray structural studies were made on micelles of three nonionic detergents: lauryl ether of polyoxyethylene(9) alcohol, lauryl ester of sucrose, and stearyl ester of sucrose. The contrast variation method was used by changing sucrose concentration in solutions. Observed X-ray intensity distribution was explained by either of two models: (1) micelles are spherical in shape and polydisperse in mass and (2) micelles are oblate ellipsoid of revolution and monodisperse. Structural parameters of micelles of the three detergents were determined for the two models. On the X-ray intensity vs s curves, where s = 2 × sin θ/λ, λ and 2θ being the X-ray wavelength and the scattering angle, there are specific s at which the intensity is practically independent of the electron density of solvent. Such s are called an isoscattering point and denoted as s∗ . Theoretically s∗ is given by 2πs∗R 0 = tan (2πs∗R 0 ) , if all micelles are identical, having a spherical shape function of radius R0. The isoscattering point is useful to determine micelle volume even though the micelles are neither spherical nor monodisperse, if the deviation from spherical shape and monodispersity is not large.

Effects of volatile anesthetics on bacteriorhodopsin in purple membrane, Halobacterium halobium cells and reconstituted vesicles

In this study, we have investigated effects of volatile anesthetics on absorption spectra, proton pumping activity and decay of photointermediate M of bacteriorhodopsin (bR) in differently aggregated states. Anesthetics used in this study are ether-type general anesthetics; enflurane and sevoflurane. The observed effects on bR depend not only on variety or concentration of anesthetics but also strongly on the aggregation state of bR molecules in the membrane. In purple membrane (PM), bR having maximum light absorption at 567 nm (bR567) is formed in the presence of sevoflurane or a small amount of enflurane, while a species absorbing maximally at 480 nm (bR480) is formed upon the addition of large amounts of enflurane. X-ray diffraction studies show that the former species maintains crystallinity of PM, but the latter does not. In reconstituted vesicles where bR molecules exist as monomer, even sevoflurane forms bR480. Flash photolysis experiments show that bR567 contains a shorter-lived M intermediate absorbing maximally at 412 nm in the photoreaction cycle than bR does and that bR480 contains at least two long-lived M intermediates which seem to absorb maximally near and at lower than 380 nm. The measurements of light-induced pH changes of the whole cells and of the reconstituted vesicles in the presence of the anesthetics indicate that bR567 has a enhanced proton pumping efficiency, while bR480 has a quite low or no activity. No significant difference was observed in the anesthetic action between two inversely pumping vesicles. These observations suggest that on the formation of bR480, anesthetics enter into the membrane and affect the protein-lipid interaction.

On some physicochemical properties of sucrose esters and the stability they confer to membrane proteins

Abstract A homologous series of fatty acid monoesters of sucrose whose chain length varies from 8 to 22 carbon atoms has been examined with respect to some physicochemical properties. In this series, the critical micelle concentration is comparable to that of lysophospholipids having similar hydrophobic moiety. The behavior of a spin probe dispersed in micelles of sucrose esters and their surface pressure-area isotherms at the air/water interface indicate that, despite a relatively large hydrophilic head, they form increasingly compact structures as their chain length increases. Finally, when used as dispersing medium for the purified intrinsic protein rhodopsin, long-chain esters confer to the pigment a thermal stability which is close to that of its membrane-bound state. We conclude that these detergents may be among those best suited to purified intrinsic membrane protein studies.