Tsutomu Kouyama

Crystal structure of squid rhodopsin.

Invertebrate phototransduction uses an inositol-1,4,5-trisphosphate signalling cascade in which photoactivated rhodopsin stimulates a Gq-type G protein, that is, a class of G protein that stimulates membrane-bound phospholipase Cβ. The same cascade is used by many G-protein-coupled receptors, indicating that invertebrate rhodopsin is a prototypical member. Here we report the crystal structure of squid (Todarodes pacificus) rhodopsin at 2.5 Å resolution. Among seven transmembrane α-helices, helices V and VI extend into the cytoplasmic medium and, together with two cytoplasmic helices, they form a rigid protrusion from the membrane surface. This peculiar structure, which is not seen in bovine rhodopsin, seems to be crucial for the recognition of Gq-type G proteins. The retinal Schiff base forms a hydrogen bond to Asn 87 or Tyr 111; it is far from the putative counterion Glu 180. In the crystal, a tight association is formed between the amino-terminal polypeptides of neighbouring monomers; this intermembrane dimerization may be responsible for the organization of hexagonally packed microvillar membranes in the photoreceptor rhabdom.

Specific Damage Induced by X-ray Radiation and Structural Changes in the Primary Photoreaction of Bacteriorhodopsin.

Bacteriorhodopsin, the sole membrane protein of the purple membrane of Halobacterium salinarum, functions as a light-driven proton pump. A 3-D crystal of bacteriorhodopsin, which was prepared by the membrane fusion method, was used to investigate structural changes in the primary photoreaction. It was observed that when a frozen crystal was exposed to a low flux of X-ray radiation (5 x 10(14)photons mm(-2)), nearly half of the protein was converted into an orange species, exhibiting absorption peaks at 450 nm, 478 nm and 510 nm. The remainder retained the normal photochemical activity until Asp85 in the active site was decarboxlyated by a higher flux of X-ray radiation (10(16)photons mm(-2)). The procedure of diffraction measurement was improved so as to minimize the effects of the radiation damage and determine the true structural change associated with the primary photoreaction. Our structural model of the K intermediate indicates that the Schiff base linkage and the adjacent bonds in the polyene chain of retinal are largely twisted so that the Schiff base nitrogen atom still interacts with a water molecule located near Asp85. With respect to the other part of the protein, no appreciable displacement is induced in the primary photoreaction.

Highly Selective Separation of Rhodopsin from Bovine Rod Outer Segment Membranes Using Combination of Divalent Cation and Alkyl(thio)glucoside

The micellization process of bovine rod outer segment (ROS) membranes is investigated utilizing a series of neutral detergents. It is found that when alkyl(thio)glucosides with an appropriate hydrophilic–lipophilic balance (e.g. octylthioglucoside) are used in combination with a divalent cation, rhodopsin is selectively extracted from ROS membranes at a specific detergent‐to‐membrane ratio. This allows remarkable purification of rhodopsin by a single‐step solubilization, because the residual membranes are heavily aggregated in the presence of divalent cation and are therefore easily sedimented by low‐speed centrifugation. The absorption spectrum of the supernatant reproducibly exhibits an A280/A500 value of 1.6, an excellent value that could rarely be obtained by chromatographic purification. The degree of purification also depends on the type of divalent cation included in the solubilization solution; specific binding of IIB‐series cations (Zn2+ and Cd2+) to ROS membranes is suggested to play an important role in the solubilization process. The present result represents a unique example of selective solubilization of a specific membrane protein from highly aggregated membranes.

Specific lipid–protein interactions in a novel honeycomb lattice structure of bacteriorhodopsin

In the purple membrane of Halobacterium salinarium, bacteriorhodopsin trimers are arranged in a hexagonal lattice. When purple membrane sheets are incubated at high temperature with neutral detergent, membrane vesicularization takes place, yielding inside-out vesicles with a diameter of 50 nm. The vesicular structure becomes unstable at low temperature, where successive fusion of the vesicles yields a crystal which is composed of stacked planar membranes. X-ray crystallographic analysis reveals that the bacteriorhodopsin trimers are arranged in a honeycomb lattice in each membrane layer and that neighbouring membranes orient in opposite directions. The native structure of the trimeric unit is preserved in the honeycomb lattice, irrespective of alterations in the in-plane orientation of the trimer. One phospholipid tightly bound to a crevice between monomers in the trimeric unit is suggested to act as a glue in the formation of the trimer.

Optical monitoring of freeze-trapped reaction intermediates in protein crystals: a microspectrophotometer for cryogenic protein crystallography

A microspectrophotometer for cryogenic protein crystallography is described. It is capable of measuring visible absorption spectra (350–800 nm) of a single crystal during X-ray data collection. The microspectrophotometer is designed to minimize the level of stray light by using a double monochromator and an optical microscope equipped with two field diaphragms and a pinhole. In this system, a thick crystal with an optical density of ∼5 is measurable. In order to demonstrate the performance of the system, the absorption spectra of the unphotolyzed state and the primary photoreaction intermediate of bacteriorhodopsin in the P622 crystal have been measured under a flow of cold nitrogen gas at 100 K.

Polyhedral assembly of a membrane protein in its three-dimensional crystal

A novel ordered assemblage of bacteriorhodopsin, a transmembrane protein functioning as a light-driven proton pump, is found in its three-dimensional crystal. Atomic force microscope images of the crystal surface reveal that spherical protein clusters with a diameter of approximately 50 nm are hexagonally close-packed. Electron micrographs of mechanically disintegrated crystals show that the inside of the protein cluster is filled with the mother liquor. The crystal is made up of hollow protein clusters. When disintegrated crystals are illuminated in the presence of a lipophilic anion, a significant alkalization of the external medium occurs. This result indicates that the protein cluster contains native lipids and that the cytoplasmic side of the protein faces the external medium. X-ray diffraction patterns and the observed diameter of the spherical shell suggest that approximately 200 bacteriorhodopsin trimers are aligned on a polyhedral surface lattice. Another remarkable feature of the spherical assemblies of bacteriorhodopsin is that they fuse with each other at low ionic strength and occasionally form a tubular or doughnut-like structure. The concept of membrane protein polymorphism is introduced on the basis of these observations, and it is used to describe the dynamic structure of some other biological membranes.

Structure and function of bacteriorhodopsin

Bacteriorhodopsin (bR) is a powerful light-driven proton pump. We have developed a procedure to prepare bR-containing membrane vesicles in which a pH gradient as large as 4 pH units can be generated and maintained in the light. Using such a system, we have demonstrated that bR exhibits a high proton pump activity in a wide pH region; it works well at least between pH 4 and 9.5. For the large light-induced pH change in the external medium, the presence of a high concentration (approximately 0.1 M) of magnesium or transition metal ion is required. It is suggested that the influx of magnesium ion, electrically coupled with the proton release, takes place in the light. The three-dimensional structure of bR was studied by fluorescence energy transfer techniques. It was shown that the retinal chromophore is located 10 A below a surface of purple membrane. The in-plane location and orientation of retinal was also determined; it exists in a pocket surrounded by the helices 3, 4, 5, and 6. The position of a fluorescent probe labeled to Lys 41 was determined to be near the helix 7. Based on the results obtained, we propose a model of the bR structure. The dynamic structure of bR was investigated by fluorescence depolarization techniques. It was shown that the retinal chromophore is tightly buried in a pocket within the protein. In the presence of detergents like octylglucoside, its tertiary structure can be stable near the electric isosbestic point. The rate of dissociation/association process of bR molecules is sensitive to the pH of the medium. Dimeric and/or trimeric bR can exist stably if the concentration of detergent and other solvent conditions are adequately controlled. The photoreaction of bR in purple membrane, including the dark/light adaptation, the trans photocycle and the primary photoreaction, was reported. With respect to the trans photocycle, we found that, at alkaline pH, an M-like photoproduct (NM) is generated by excitation of a long-lived photoproduct N560 which has a major absorption maximum near 560 nm. We suggest that, at alkaline pH, the overall photoreaction of bR under steady illumination is approximated by the two-photon cycle: bR570 approximately greater than M412----N560 approximately greater than NM----bR570. With respect to the primary photoreaction, we found that the fluorescence quantum yield of the near-infrared emission of bR was greatly enhanced at acidic pH (approximately pH 2).(ABSTRACT TRUNCATED AT 400 WORDS)

Electron Cryomicroscopy of Bacteriorhodopsin Vesicles: Mechanism of Vesicle Formation

We obtained vesicles from purple membrane of Halobacterium halobium at different suspension compositions (pH, electrolytes, buffers), following the procedure of Kouyama et al. (1994) (J. Mol. Biol. 236:990-994). The vesicles contained bacteriorhodopsin (bR) and halolipid, and spontaneously formed during incubation of purple membrane suspension in the presence of detergent octylthioglucoside (OTG) if the protein:OTG ratio was 2:1 by weight. The size distribution of the vesicles was precisely determined by electron cryomicroscopy and was found to be almost independent on the incubation conditions (mean radius 17.9-19 nm). The size distribution in a given sample was close to the normal one, with a standard deviation of approximately +/- 1 nm. During dialysis for removal of the detergent, the vesicles diminished their radius by 2-2.5 nm. The results allow us to conclude that the driving force for the formation of bR vesicles is the preferential incorporation of OTG molecules in the cytoplasmic side of the membrane (with possible preferential delipidation of the extracellular side), which creates spontaneous curvature of the purple membrane. From the size distribution of the vesicles, we calculated the elasticity bending constant, K(B) approximately 9 x 10(-20) J, of the vesicle wall. The results provide some insight into the possible formation mechanisms of spherical assembles in living organisms. The conditions for vesicle formation and the mechanical properties of the vesicles could also be of interest with respect to the potential technological application of the bR vesicles as light energy converters.

Excited-state dynamics of bacteriorhodopsin

Near infrared emission of bacteriorhodopsin at neutral pH and at room temperature was characterized by a large Stokes shift. This characteristic was lost in an acidic pH (approximately pH 2) where a remarkable enchancement (more than 10 times) in the fluorescence quantum yield accompanied the red shift in the main absorption band. It is suggested from fluorescence polarization measurements that the emission occurs from the first allowed excited state of the retinylidene chromophore, irrespective of pH. We suggest that the large Stokes shift observed at neutral pH is a result of a charge displacement (e.g., proton translocation) that occurs immediately after excitation, and is prevented by protonation (in the ground state) of an amino-acid residue in the protein.

Fluorescence study of ϵ‐ADP bound to rabbit F‐actin: Structural change in the adenine subsite of F‐actin under the influence of heavy meromyosin

In a recent nanosecond pulse-fluorometric study of Factin+ADP [ 11, it was found that e-ADP is tightly bound in F-actin and the macromolecular motion of Factin is characterised by a correlation time as long as several microseconds. The study is extended in the present work so that the fluorescence of e-ADP bound to F-actin was measured in the presence of an Increasing amount of heavy meromyosin. Both static and time-dependent fluorescence measurements show that binding of heavy meromyosin to F-actin induces a cooperative structural change in the adenine subsite of F-actin. The cooperativity is enhanced by the combination of F-actin-e-ADP with tropomyosin.