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F Tokunaga

Publications and source records attributed to F Tokunaga.

At least 55 records · Page 3Linked to original sources
PubMed1997

Evidence for proton transfer from Glu-46 to the chromophore during the photocycle of photoactive yellow protein.

Photoactive yellow protein (PYP) belongs to the novel group of eubacterial photoreceptor proteins. To fully understand its light signal transduction mechanisms, elucidation of the intramolecular pathway of the internal proton is indispensable because it closely correlates with the changes in the hydrogen-bonding network, which is likely to induce the conformational changes. For this purpose, the vibrational modes of PYP and its photoproduct were studied by Fourier transform infrared spectroscopy at -40 degrees C. The vibrational modes characteristic for the anionic p-coumaryl chromophore (Kim, M., Mathies, R. A., Hoff, W. D., and Hellingwerf, K. J. (1995) Biochemistry 34, 12669-12672) were observed at 1482, 1437, and 1163 cm-1 for PYP. However, the bands corresponding to these modes were not observed for PYPM, the blue-shifted intermediate, but the 1175 cm-1 band characteristic of the neutral p-coumaryl chromophore was observed, indicating that the phenolic oxygen of the chromophore is protonated in PYPM. A 1736 cm-1 band was observed for PYP, but the corresponding band for PYPM was not. Because it disappeared in the Glu-46 --> Gln mutant of PYP, this band was assigned to the C=O stretching mode of the COOH group of Glu-46. These results strongly suggest that the proton at Glu-46 is transferred to the chromophore during the photoconversion from PYP to PYPM.

Bacterial Proteins↗
PubMed1997

A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin.

During light-driven proton transport bacteriorhodopsin shuttles between two protein conformations. A large-scale structural change similar to that in the photochemical cycle is produced in the D85N mutant upon raising the pH, even without illumination. We report here that (i) the pKa values for the change in crystallographic parameters and for deprotonation of the retinal Schiff base are the same, (ii) the retinal isomeric configuration is nearly unaffected by the protein conformation, and (iii) preventing rotation of the C13-C14 double bond by replacing the retinal with an all-trans locked analogue makes little difference to the Schiff base pKa. We conclude that the direct cause of the conformational shift is destabilization of the structure upon loss of interaction of the positively charged Schiff base with anionic residues that form its counter-ion.

Bacteriorhodopsins↗
PubMed1997

Functional expression and characterization of frog photoreceptor-specific calcium-binding proteins.

S-modulin (sensitivity-modulating protein) is a photoreceptor-specific calcium-binding protein which plays an important role in the light adaptation process by controlling rhodopsin phosphorylation in rods. S-modulin and its cone homologue, s26, were expressed at high level (more than 30% of total protein) in Escherichia coli and then purified. They both inhibited rhodopsin phosphorylation in a calcium dependent manner. Myristoylated recombinants of S-modulin and s26 showed calcium-dependent changes in tryptophan emission spectra with half-maxima at about 0.7 microM free calcium concentration. However, the spectral changes are distinctive from each other, suggesting that there is some difference in the structural change between S-modulin and s26.

Adenosine Triphosphate↗
PubMed1997

Rab proteins of Drosophila melanogaster: novel members of the Rab-protein family.

From a Drosophila head cDNA library, we isolated 9 cDNA clones, each of which encodes a different member of Rab-protein family. Seven of them (DRabs) have more than 80% amino acid identity to the corresponding members of mammalian Rab proteins. The other two proteins (DRabRP3 and 4) show low sequence similarity to any of the known Rab proteins. However, both contain all amino acids conserved in known Rabs. In addition, DRabRP4 has strong GTP-binding activity, when synthesized in E. coli cells. These results indicate that DRabRPs are novel members of the Rab-protein family. Molecular phylogenetic analysis also supported this conclusion.

Amino Acid Sequence↗
PubMed1997

Structural characterization of the molten globule of alpha-lactalbumin by solution X-ray scattering.

A compact denatured state is often observed under a mild denaturation condition for various proteins. A typical example is the alpha-lactalbumin molten globule. Although the molecular compactness and shape are the essential properties for defining the molten globule, there have been ambiguities of these properties for the molten globule of alpha-lactalbumin. Using solution X-ray scattering, we have examined the structural properties of two types of molten globule of alpha-lactalbumin, the apo-protein at neutral pH and the acid molten globule. The radius of gyration for the native holo-protein was 15.7 A, but the two different molten globules both had a radius of gyration of 17.2 A. The maximum dimension of the molecule was also increased from 50 A for the native state to 60 A for the molten globule. These values clearly indicate that the molten globule is not as compact as the native state. The increment in the radius of gyration was less than 10% for the alpha-lactalbumin molten globule, compared with up to 30% for the molten globules of other globular proteins. Intramolecular disulfide bonds restrict the molecular expansion of the molten globule. The distance distribution function of the alpha-lactalbumin molten globule is composed of a single peak suggesting a globular shape, which is simply swollen from the native state. The scattering profile in the high Q region of the molten globule indicates the presence of a significant amount of tertiary fold. Based on the structural properties obtained by solution X-ray scattering, general and conceptual structural images for the molten globules of various proteins are described and compared with the individual, detailed structural model obtained by nuclear magnetic resonance.

Animals↗
PubMed1997

The primary structure and distribution of killifish visual pigments.

Five cDNA fragments (KFH-R, -V, -G, -B and -Rh) encoding the putative visual pigments of killifish were isolated and sequenced. Judging from the deduced amino acid sequences, each cDNA falls into a different group of the five major families of vertebrate visual pigment genes. In situ hybridization localized the mRNA of KFH-R and -G to the principle and accessory members, respectively, of double cones. Visual pigment genes KFH-Rh, -B and -V were expressed in the rods, and the long and short single cones, respectively. It is suggested that the relationships between the cell types and their respective visual pigment gene groups may be a common pattern among teleost fishes.

Amino Acid Sequence↗
PubMed1997

Functional expression and site-directed mutagenesis of photoactive yellow protein.

The gene encoding photoactive yellow protein (PYP) was isolated from Ectothiorhodospira halophila, and a high-level expression system for PYP was constructed in Escherichia coli. The molecular weight and the absorption spectrum of PYP expressed in E. coli were identical with those of the native PYP isolated from E. halophila. The amino acid residues which might interact with the chromophore (Tyr42, Glu46, Thr50, Arg52, and Cys69) were mutated by site-directed mutagenesis and the absorption spectra of these mutants were examined to study the chromophore/protein interaction in PYP. The former three substitutions (Y42F, E46Q, and T50V) brought about red-shifts of the absorption spectra, but the substitution of Arg52 (R52Q) brought about no change and that of Cys69 (C69S) led to no formation of pigments. These results suggest that Tyr42, Glu46, and Thr50 strongly interact with the chromophore, while Arg52 does not contribute the color tuning of PYP.

Amino Acid Sequence↗
PubMed1997

X-ray diffraction studies of bacteriorhodopsin. Determination of the positions of mercury label at several engineered cysteine residues.

The single cysteine-containing bacteriorhodopsin mutants F27C, L100C, T170C, F171C and I222C were labeled with p-chloromercuribenzoic acid, which specifically reacts with sulfhydryl groups. These cysteines should be located at the cytoplasmic ends of the transmembrane helices A, C, F or G. We determined the positions of the bound mercury atoms by X-ray diffraction of purple membrane films, with better than 1 A accuracy. The determined mercury positions were compared with the structural model from cryoelectron microscopy (N. Grigorieff, T. A. Ceska, K. H. Downing, J. M. Baldwin and R. Henderson, J. Mol. Biol. 259, 393-421, 1996). Given that the distance between the mercury and the C alpha atom of the cysteine in the xy plane must be shorter than 4.5 A and that the mercury atom is located at the delta position, the positions obtained for the mercury labels agree with their expected positions from the structural model. The present results give a rationale for detecting structural changes upon illumination as shifts occur in the mercury label position.

Amino Acid Sequence↗
PubMed1997

Characterization of lamprey rhodopsin by isolation from lamprey retina and expression in mammalian cells.

A visual pigment was extracted from lamprey retina and was expressed in cultured mammalian cells (293S) using a cDNA fragment isolated from lamprey retina. The extracted pigment, a putative lamprey rhodopsin, had an absorption maximum at 503 nm. The recombinant lamprey rhodopsin, reconstituted with 11-cis-retinal, showed an absorption maximum at about 500 nm. Both pigments reacted with an anti-bovine rhodopsin antibody (Rh29), which recognizes the short photoreceptor cells in lamprey retina. Unlike rhodopsins of higher vertebrates, the lamprey rhodopsin bleached gradually in the presence of 100 mM hydroxylamine even in the dark. Our results suggest that, despite its high similarities with other vertebrate rhodopsins, lamprey rhodopsin has a character different from those of higher vertebrates.

Animals↗
PubMed1997

In situ inhibition of vesicle transport and protein processing in the dominant negative Rab1 mutant of Drosophila.

Rab proteins play an essential role in vesicle transport. In particular, RAB1 is thought to participate in the transport of most membrane and secretory proteins. To investigate the role of RAB1 in developing or functioning cells in situ, we constructed transgenic, dominant-negative Rab1 mutants of Drosophila, and examined the protein transport and cellular and subcellular structures of mutant photoreceptor cells. In the transgenic fly, the expression of mutant RAB1 was induced by Gal4 protein, whose expression was triggered by heat treatment (37 degrees C) of the fly. Within several hours after the heat induction, the lumens of the rough endoplasmic reticulum (rER) became swollen, and Golgi bodies were disassembled into vesicle clusters. Corresponding to these changes in cell structure, rhodopsin transport was blocked between the rER and the Golgi body, as indicated by the accumulation of immature rhodopsin carrying a large high-mannose-type oligosaccharide chain. Long-term expression of mutant RAB1 caused the degradation of photoreceptive microvilli and the accumulation of numerous swollen rERs, whereas no distinct changes were found in the axonal regions. These results indicate that, in Drosophila photoreceptor cells, RAB1 contributes to the maintenance of local cell structure by mediating vesicle transport between the rER and Golgi body.

Amino Acid Substitution↗
PubMed1996

Limulus factor D, a 43-kDa protein isolated from horseshoe crab hemocytes, is a serine protease homologue with antimicrobial activity.

A glycoprotein (Mr = 43,000) from horseshoe crab hemocytes with antimicrobial activity against Gram-negative bacteria was purified. The internal peptide sequences coincided exactly with the deduced amino acid sequence of a cDNA clone, designated limulus factor D, which was isolated by screening a hemocyte cDNA library with an anti-human plasminogen antibody. The open reading frame codes for a precursor of factor D of 394 amino acid residues, including an NH2-terminal signal sequence. The COOH-terminal domain of factor D has significant sequence homology with the catalytic domain of mammalian serine proteases, in particular with human tissue plasminogen activator (32% identity), except for the substitution of Ser of the active site triad to Gly. Factor D has a unique NH2-terminal domain with weak sequence homology with part of the mammalian interleukin-6 receptor alpha-chain. Factor D is likely to have an important role in host defense mechanisms.

Amino Acid Sequence↗
PubMed1996

Photoreaction cycle of photoactive yellow protein from Ectothiorhodospira halophila studied by low-temperature spectroscopy.

The photocycle of photoactive yellow protein (PYP) from Ectothiorhodospira halophila was studied by low-temperature spectroscopy. Irradiation of PYP at -190 degrees C produced a photo-steady-state mixture composed of bathochromic and hypsochromic photoproducts (PYPB and PYPH). Upon warming, PYPH was thermally converted to a slightly blue-shifted intermediate (PYPHL) above -150 degrees C and then to a red-shifted one (PYPL) above -80 degrees C. PYPB was thermally converted to the blue-shifted intermediate (PYPBL) above -180 degrees C and then to PYPL above -90 degrees C. PYPL thermally reverted to PYP above -50 degrees C, completing the photocycle. The spectral properties of PYPL formed at low temperature suggest that it corresponds to the red-shifted photoproduct detected in the nano- to microsecond time scale at room temperature (A465). The absolute absorption spectra of PYPH, PYPB, and PYPL were estimated, and their absorption maxima were determined to be 442 and 489 nm at -190 degrees C and 456 nm at -80 degrees C, respectively. Although a near-UV intermediate (A355) is observed in the recovery process of PYP from A465 at room temperature, it was not detected at low temperatures, probably due to the effects of temperature and the presence of glycerol. A scheme of the photocycle of PYP is presented.

Bacteria↗
PubMed1996

Photoreceptor protein s26, a cone homologue of S-modulin in frog retina.

A frog retinal protein named s26 is a 26-kDa protein found during purification of S-modulin in frog retina (Kawamura, S. (1992) Photochem. Photobiol. 56, 1173-1180). To identify its role in frog retina, first s26 was purified to nearly homogeneity with three chromatographical steps. Based on the partial amino acid sequences of the proteolysed fragments of s26, we isolated cDNAs that encode s26. The analysis of its amino acid sequence revealed that s26 is an S-modulin-like protein, while it shows higher homology to visinin. Visinin is a Ca2+-binding protein reported to be present in chicken cones, but its localization in the retina had been a subject in dispute. The present study showed that s26 is present in cone photoreceptors. The study also showed that s26 inhibits phosphorylation of rhodopsin after a light flash at high Ca2+ concentrations as S-modulin does. From these results, we concluded that s26 is a cone homologue of S-modulin. The result is consistent with the idea that each type of photoreceptors expresses each cell-type specific version of phototransduction proteins.

Amino Acid Sequence↗
PubMed1996

Molecular properties of chimerical mutants of gecko blue and bovine rhodopsin.

In spite of the high similarity in amino acid sequence between rod visual pigment rhodopsin and gecko blue-sensitive pigment (gecko blue), not only the spectral sensitivities but also the thermal decay rates of the meta II- and III-intermediates are noticeably different from one another [Kojima et al. (1995) Biochemistry 34, 1096-1106]. In order to identify the protein region(s) that contain(s) key residue being responsible for the functional difference, we constructed six chimerical mutants derived from gecko blue and bovine rhodopsin, with the aid of protein production in a human embryonic kidney cell line (293S). While the absorption maximum of every mutant was located in between gecko blue (466 nm) and bovine rhodopsin (500 nm), a large blue-shift (18 nm) was observed when the helices I-III of rhodopsin were replaced with those of gecko blue. A time resolved spectroscopic study demonstrated that this replacement also accelerated the decay rate of the meta II-intermediate. The decay of the meta III-intermediate of the mutants became faster as the compartment of gecko blue was increased. Thus, the faster decay of the meta II-intermediate of gecko blue is largely attributed to residues within helices I-III, while the decay of the meta III-intermediate apparently depends on the overall structure of the protein.

Animals↗
PubMed1996

Structure of the N intermediate of bacteriorhodopsin revealed by x-ray diffraction.

X-ray diffraction experiments revealed the structure of the N photointermediate of bacteriorhodopsin. Since the retinal Schiff base is reprotonated from Asp-96 during the M to N transition in the photocycle, and Asp-96 is reprotonated during the lifetime of the N intermediate, or immediately after, N is a key intermediate for understanding the light-driven proton pump. The N intermediate accumulates in large amounts during continuous illumination of the F171C mutant at pH 7 and 5 degrees Celsius. Small but significant changes of the structure were detected in the x-ray diffraction profile under these conditions. The changes were reversible and reproducible. The difference Fourier map indicates that the major change occurs near helix F. The observed diffraction changes between N and the original state were essentially identical to the diffraction changes reported for the M intermediate of the D96N mutant of bacteriorhodopsin. Thus, we find that the protein conformations of the M and N intermediates of the photocycle are essentially the same, in spite of the fact that in M the Schiff base is unprotonated and in N it is protonated. The observed structural change near helix F will increase access of the Schiff base and Asp-96 to the cytoplasmic surface and facilitate the proton transfer events that begin with the decay of the M state.

Aspartic Acid↗
PubMed1996

Molecular and cellular basis for type I heparin cofactor II deficiency (heparin cofactor II Awaji).

Heparin cofactor II (HCII) is a serine proteinase inhibitor in human plasma that rapidly inhibits thrombin in the presence of dermatan sulfate or heparin. To understand the molecular mechanism for HCII deficiency in a patient with reduced circulating HCII antigen, we studied a Japanese patient with type I HCII deficiency who suffered from angina pectoris and coronary artery disease. Polymerase chain reaction (PCR)-based sequence analysis showed that the propositus' gene for HCII (HCII Awaji gene) had a thymine insertion after codon (GAT) for Asp88 in exon II, resulting in a frameshift mutation. Consequently, the abnormal HCII Awaji protein was suggested to have an altered amino acid sequence from position 89 and terminate at 107, thus being composed of the NH2-terminal one fifth of normal HCII and dysfunctional for thrombin inhibition. The molecular weight and pI value of HCII Awaji were calculated to be 12,040 and 3.6, respectively, without posttranslational modification. Mutagenic PCR followed by the Tsp509I digestion showed that a half of the PCR products derived from the propositus and his sister was cleaved, suggesting that his sister also has the same mutant allele. Crossed-immunoelectrophoresis and Western blot analyses of plasma and urine from the the propositus and of plasma from his sister did not provide evidence for the existence of the abnormal HCII, suggesting that little truncated HCII was circulating in the patient's blood. However, stable expression assay using human kidney 293 cells transfected with the expression vector containing cDNA encoding wild-type or Awaji-type HCII showed that mutant as well as wild-type HCII was secreted into culture medium normally. These results suggest that the abnormal HCII Awaji protein is secreted normally, but rapidly degraded in the circulating blood.

Amino Acid Sequence↗
PubMed1996

The linker of calmodulin lacking Glu84 is elongated in solution, although it is bent in the crystal.

The solution structure of a mutant calmodulin (des84) lacking Glu84 in the central helix linking the two calmodulin lobes is substantially different from its crystal structure. As determined by small-angle X-ray scattering, the radius of gyration and the maximum linear dimension of des84 in the presence of 0.1 mM calcium are 20.8 A and 62.5 A, respectively. These respective dimensions are larger than those expected from the crystal structure of des84, 18.5 A and 55.0 A, and smaller than those expected from the crystal structure of wild type, 22.8 A and 67.5 A. The distance distribution function of des84 indicates that it assumes an elongated, dumbbell shape in solution. The solution scattering profile of des84 is indistinguishable from that of wild-type calmodulin. The calcium-dependent binding of melittin to des84 causes a change in its shape from elongated to spherical, as seen with other calmodulins. In comparison with calcium-saturated des84, calcium-free des84 is slightly elongated; a slight compaction is observed with native calmodulin. The observed differences between the averaged solution structure and the crystal structure of des84 suggests that an ensemble of structures is available to calmodulin in solution and that its target need not induce a change in its conformation. These results support the theory that the linker region of the central helix of calmodulin functions as a flexible tether.

Calcium↗