Biomedical subjects

Fumio Tokunaga

Publications and source records attributed to Fumio Tokunaga.

At least 19 recordsLinked to original sources

PubMed2006

Induced expression of hematopoietic- and neurologic-expressed sequence 1 in retinal pigment epithelial cells during newt retina regeneration.

Newts can regenerate their organs even as adults. For instance, when their neural retinas are completely removed by operation, the remaining retinal pigment epithelial (RPE) cells dedifferentiate to reconstruct neural retinas. To elucidate the molecular mechanisms of newt retina regeneration, we investigated genes upregulated in dedifferentiating RPE cells using differential display methods. We observed that a cDNA fragment of hematopoietic- and neurologic-expressed sequence 1 (Hn1) appeared to be induced within a few days of surgical removal of newt neural retina. Using an anti-HN1 antiserum against the recombinant HN1 protein, we carried out immunohistochemical analyses. The anti-HN1 antiserum recognized the plexiform layers and ganglion cell layer (GCL) but not the RPE cell layer in unoperated (normal) newt retinas. Using a glial fibrillary acidic protein antibody, Hn1 was shown to be possibly expressed in glial cells in normal neural retina. During retina regeneration, immunoreactivity for HN1 appeared in dedifferentiating RPE cells 10 days post-operation, and in retinal progenitor cells 18 days post-operation. Twenty seven days post-operation, HN1 immunoreactivity was localized in the plexiform layers and GCL as in the normal retina. Therefore, HN1 possibly plays an undefined role in dedifferentiating RPE cells and retinal progenitor cells during newt retina regeneration.

Amino Acid Sequence↗

PubMed2006

Time-resolved thermodynamics: heat capacity change of transient species during photoreaction of PYP.

Heat capacity changes of short-lived transient species in different time ranges were measured for the first time by using the thermal component of the transient grating and transient lens signals at various temperatures. This method was applied to the transient intermediates of Photoactive Yellow Protein (PYP). The temperature dependence of the enthalpy change shows that the heat capacity of the short-lived intermediate pR2 (also called I1 or PYP(L)) species is the same as that of the ground state (pG) species within our experimental accuracy, whereas that of the long-lived intermediate pB (I2 or PYP(M)) is much larger (2.7 +/- 0.4 kJ/mol K) than that of pG. The larger heat capacity is interpreted in terms of the conformational change of the pB species such as melted conformation and/or exposure of the nonpolar residues to the aqueous phase. This technique can be used for photochemical reactions in general to investigate the conformational change and the hydrophobic interaction in a time domain.

Bacterial Proteins↗

PubMed2005

Structural changes during the photocycle of photoactive yellow protein monitored by ultraviolet resonance raman spectra of tyrosine and tryptophan.

Photoactive yellow protein (PYP) is a bacterial blue light photoreceptor, and photoexcitation of dark-state PYP (PYP(dark)) triggers a photocycle that involves several intermediate states. We report the ultraviolet resonance Raman spectra of PYP with 225-250 nm excitations and investigate protein structural changes accompanying the formation of the putative signaling state denoted PYP(M). The PYP(M)-PYP(dark) difference spectra show several features of tyrosine and tryptophan, indicating environmental changes for these amino acid residues. The tyrosine difference signals show small upshifts with intensity changes in Y8a and Y9a bands. Although there are five tyrosine residues in PYP, Tyr42 and Tyr118 are suggested to be responsible for the difference signals on the basis of a global fitting analysis of the difference spectra at different excitation wavelengths and the crystal structure of PYP(dark). A further experiment on the Thr50-->Val mutant supports environmental changes in Tyr42. The observed upshift of the Y8a band suggests a weaker or broken hydrogen bond between Tyr42 and the chromophore in PYP(M). In addition, a reorientation of the OH group in Tyr42 is suggested from the upshift of the Y9a band. For tryptophan, the Raman bands of W3, W16, and W18 modes diminish in intensity upon formation of PYP(M). The loss of intensities is attributable to an exposure of tryptophan in PYP(M). PYP contains only one tryptophan (Trp119) that is located more than 10 A from the active site. Thus the observed changes are indicative of global conformational changes in protein during the transition from PYP(dark) to PYP(M). These results are in line with the currently proposed photocycle mechanism of PYP.

Bacterial Proteins↗

PubMed2005

A biosensor in the time domain based on the diffusion coefficient measurement: intermolecular interaction of an intermediate of photoactive yellow protein.

A new type of biosensor is presented for the first time. This method is based on the diffusion measurement in the time domain by the transient grating method. As the first demonstration of this new method, various intermolecular interactions with a reaction intermediate of photoactive yellow protein, such as the protein-protein interaction, protein-DNA interaction, and protein-small molecule binding are detected. The characteristic advantages and limitations are summarized.

Bacterial Proteins↗

PubMed2005

Downregulation of Otx2 in the dedifferentiated RPE cells of regenerating newt retina.

Cynops pyrrhogaster (the Japanese common newt) regenerates neural retina from retinal pigmented epithelium (RPE) cells. Otx2 is a transcription factor that is involved in RPE cell differentiation. To understand the role of Otx2 during transdifferentiation of RPE cells, we cloned a Cynops Otx2 cDNA, and explored its expression by RT-PCR, immunohistochemistry and in situ hybridization. The expression of Otx2 was compared with the localization of a proliferating cell marker (PCNA), RPE cell markers (RPE65, CRBP) and an RPE and Muller glial cell marker (CRALBP). At the early stage of regeneration, 2 to 3 cell layered regenerating retina consisting of pigmented cells uniformly expressed Otx2 and other markers. Following this stage, 4-cell layered regenerating retina consisted of two distinct layers, pigmented monolayer (the outer layer) attached to Bruch's membrane and presumptive neural retina (the inner layers). In the outer layer, Otx2 and CRBP expression was maintained and majority of cells lost PCNA expression. Some of cells maintained RPE65. In the inner layers, expression of Otx2, CRBP and RPE65 was downregulated, but a majority of those cells maintained PCNA expression. These results indicate that spatiotemporal regulation of Otx2 expression is consistent with those of RPE markers. Otx2 may play a pivotal role in maintenance and specification of RPE cells during neural retina regeneration. In contrast to RPE cell markers, CRALBP was expressed in both the pigmented and the de-pigmented layers. This observation implicates the appearance of Muller glial cells in an early phase of regenerating retina.

Animals↗

PubMed2004

Structure of photoactive yellow protein (PYP) E46Q mutant at 1.2 A resolution suggests how Glu46 controls the spectroscopic and kinetic characteristics of PYP.

Photoactive yellow protein from Ectothiorhodospira halophila is a photoreceptor protein involved in the negative phototaxis of this bacterium. Its chromophore (p-coumaric acid) is deprotonated in the ground state, which is stabilized by a hydrogen-bond network between Tyr42, Glu46 and Thr50. Glu46 is a key residue as it has been suggested that the proton at Glu46 is transferred to the chromophore during its photoconversion from the dark state to the signalling state. The structure of E46Q mutant protein was determined at 1.2 A resolution, revealing that the phenolic O atom of p-coumaric acid is hydrogen bonded to NH(2) of Gln46 in E46Q with a longer distance (2.86 +/- 0.02 A) than its distance (2.51 A) to Glu46 OH in the wild type. This and the decreased thermal stability of E46Q relative to the wild type show that this hydrogen bond is weakened in the E46Q mutant compared with the corresponding bond in the wild type. Several characteristic features of E46Q such as an alkali shift in the pK(a) and the rapid photocycle can be explained by this weakened hydrogen bond. Furthermore, the red shift in the absorption maximum in E46Q can be explained by the delocalization of the electron on the phenolic oxygen of p-coumaric acid owing to the weakening of this hydrogen bond.

Bacterial Proteins↗

PubMed2004

Phosphorylation of teleost phosducins and its effect on the affinity to G-protein beta gamma subunits.

Phosducin (PD) is a regulatory protein involved in the phototransduction cascade of vertebrate photoreceptor cells. We have previously demonstrated that there are rod- and cone-specific PDs (OlPD-R and OlPD-C) in the retina of the teleost fish, medaka (Oryzias latipes) [FEBS Lett. 502 (2001) 117]. A 6x His affinity precipitation assay revealed that phosphorylation by either protein kinase A (PKA) or Ca(2+)/calmodulin-dependent kinase II (CaMKII) reduced the affinity of recombinant medaka PDs to endogenous medaka G-protein beta gamma subunits (Gbetagamma). These results suggest that the affinity of medaka PDs to Gbetagamma is regulated by cAMP and Ca(2+) concentrations as also found for mammalian PDs. However, we found a specific difference in the phosphorylation patterns between recombinant OlPD-R and OlPD-C, which resulted in different affinities to Gbetagamma. These differences may affect the light/dark-adaptation between medaka rods and cones.

Animals↗

PubMed2004

Resonance Raman evidence for two conformations involved in the L intermediate of photoactive yellow protein.

The blue light receptor photoactive yellow protein (PYP) displays a photocycle that involves several intermediate states. Here we report resonance Raman spectroscopic investigations of the short-lived red-shifted intermediate denoted PYP(L). We have found that the Raman bands of the carbonyl C=O stretching mode nu(11) as well as the C=C stretching mode nu(13) for the chromophore can be resolved into two peaks, and the ratio of the two components varies as a function of pH with pK(a) approximately 6. The isotope effects on the resonance Raman spectra have confirmed a deprotonated cis-chromophore for the two components. The results indicate the presence of two conformations in the active site of PYP(L). The normal coordinate calculations based on the density functional theory provide a structural model for the two conformations, where the low pH form is possibly an active structure for the protonation reaction generating a following intermediate in the photocycle.

Bacterial Proteins↗

PubMed2004

Resonance Raman spectroscopy reveals the origin of an intermediate wavelength form in photoactive yellow protein.

Photoactive yellow protein (PYP) is a bacterial blue light receptor containing a 4-hydroxycinnamyl chromophore, and its absorption maximum is 446 nm. In a dark state, the hydroxyl group of the chromophore is deprotonated and forms hydrogen bonds with Tyr42 and Glu46. Either removal of a hydrogen bond with Tyr42 or addition of chaotropes such as thiocyanate produces a blue-shifted species called an intermediate wavelength form, in which absorption maximum ranges from 355 to 400 nm. To examine the structural origin of the intermediate wavelength form, we have performed resonance Raman investigations of wild-type PYP and some mutants (Tyr42 --> Ala, Tyr42 --> Phe, Glu46 --> Gln, and Thr50 --> Val) in the presence or absence of potassium thiocyanate. These studies show that the chromophore of the intermediate wavelength form is protonated, implying an increase in a pK(a) of the chromophore. Hence, the removal of the hydrogen bond between Tyr42 and chromophore or partial protein denaturation in the presence of thiocyanate results in a spectral blue-shift. Quantum chemical calculations based on density functional theory further support the idea that the pK(a) of the chromophore is increased by removing a hydrogen bond or by increasing the dielectric constant in the vicinity of the chromophore.

Alanine↗

PubMed2004

Time-resolved detection of sensory rhodopsin II-transducer interaction.

The dynamics of protein conformational change of Natronobacterium pharaonis sensory rhodopsin II (NpSRII) and of NpSRII fused to cognate transducer (NpHtrII) truncated at 159 amino acid sequence from the N-terminus (NpSRII-DeltaNpHtrII) are investigated in solution phase at room temperature by the laser flash photolysis and the transient grating methods in real time. The diffusion coefficients of both species indicate that the NpSRII-DeltaNpHtrII exists in the dimeric form in 0.6% dodecyl-beta-maltopyranoside (DM) solution. Rate constants of the reaction processes in the photocycles determined by the transient absorption and grating methods agree quite well. Significant differences were found in the volume change and the molecular energy between NpSRII and NpSRII-DeltaNpHtrII samples. The enthalpy of the second intermediate (L) of NpSRII-DeltaNpHtrII is more stabilized compared with that of NpSRII. This stabilization indicates the influence of the transducer to the NpSRII structure in the early intermediate species by the complex formation. Relatively large molecular volume expansion and contraction were observed in the last two steps for NpSRII. Additional volume expansion and contraction were induced by the presence of DeltaNpHtrII. This volume change, which should reflect the conformational change induced by the transducer protein, suggested that this is the signal transduction process of the NpSRII-DeltaNpHtrII.

Archaeal Proteins↗

PubMed2002

Opsin expression in adult, developing, and regenerating newt retinas.

Japanese common newts (Cynops pyrrhogaster) have an ability to regenerate their neural retina even as adults. Although extensive research has been carried out attempting to understand this retinal regeneration, the molecules characterized in newt retina are limited. We isolated cDNAs encoding three putative opsins (Cp-Rh, -LWS and -SWS1), in addition to Cp-SWS2 [Takahashi et al., FEBS Lett. 501 (2001) 151-155] from a cDNA library of adult newt retina. Our immunohistochemical and in situ hybridization studies demonstrated that Cp-Rh is selectively expressed in rods, whereas the other opsins are expressed in cones. The distribution of opsin mRNAs in normal and regenerated retinas is very similar. In both developing and regenerating retinas, Cp-Rh and its mRNA first appeared in immature rods at the beginning or just after the formation of plexiform layers. Cp-Rh was initially found isotropically in the plasma membrane, and then translocalized to the apical region along with the maturation of regenerating rods. This suggests that reorganization of the intracellular structure takes place during maturation of the regenerating newt photoreceptors.

Amino Acid Sequence↗

PubMed2002

Resonance Raman spectroscopy and quantum chemical calculations reveal structural changes in the active site of photoactive yellow protein.

Photoactive yellow protein (PYP) is a bacterial photoreceptor containing a 4-hydroxycinnamyl chromophore. Photoexcitation of PYP triggers a photocycle that involves at least two intermediate states: an early red-shifted PYP(L) intermediate and a long-lived blue-shifted PYP(M) intermediate. In this study, we have explored the active site structures of these intermediates by resonance Raman spectroscopy. Quantum chemical calculations based on a density functional theory are also performed to simulate the observed spectra. The obtained structure of the chromophore in PYP(L) has cis configuration and no hydrogen bond at the carbonyl oxygen. In PYP(M), the cis chromophore is protonated at the phenolic oxygen and forms the hydrogen bond at the carbonyl group. These results allow us to propose structural changes of the chromophore during the photocycle of PYP. The chromophore photoisomerizes from trans to cis configuration by flipping the carbonyl group to form PYP(L) with minimal perturbation of the tightly packed protein interior. Subsequent conversion to PYP(M) involves protonation on the phenolic oxygen, followed by rotation of the chromophore as a whole. This large motion of the chromophore is potentially correlated with the succeeding global conformational changes in the protein, which ultimately leads to transduction of a biological signal.

Bacterial Proteins↗

PubMed2002

Themodynamic and transport properties of intermediate states of the photocyclic reaction of photoactive yellow protein.

Themodynamic and transport properties of intermediate states of the photocyclic reaction of photoactive yellow protein (PYP) were studied by a combination of the pulsed laser-induced transient grating (TG), transient lens (TrL), and photoacoustic (PA) spectroscopies from tens of nanoseconds to hundreds of milliseconds. The diffusion coefficients (D) of PYP in the ground state (pG) and of the second intermediate state (pB) were determined by the TG analysis, and it was found that D of pG is about 1.2 times larger than D of pB. At the same time, D at various denatured conditions were measured using guanidine hydrochloride as the denaturant. D of completely unfolded protein is about 0.4 times that of the native form. The enthalpy of pB is estimated to be 60 kJ/mol by the TrL method with an assumption that the volume change of pB is not sensitive to the temperature. Since the enthalpy of the first intermediate state (pR) is as high as 160 kJ/mol, it implies that most of the photon energy is stored as the strain of the protein in pR, and this may be the driving force for the successive reaction to pB. From the temperature dependence of the volume change, the difference in the thermal expansion coefficients between pG and pR was calculated. All of the characteristic features of PYP, the negative volume change, the larger thermal expansion coefficient, and the slower diffusion process, indicate that the intermediate pR and pB are reasonably interpreted in terms of the unfolded (loosened) protein structure.

Bacterial Proteins↗

PubMed2002

Light-induced unfolding of photoactive yellow protein mutant M100L.

Light-activation of the PAS domain protein photoactive yellow protein (PYP) is believed to trigger a negative phototactic response in the phototropic bacterium Halorhodospira halophila. To investigate transient conformational changes of the PYP photocycle, we utilized the PYP mutant M100L that displays an increased lifetime of the putative signaling-state photointermediate PYP(M) by 3 orders of magnitude, as previously reported for the M100A mutant [Devanathan, S., Genick, U. K., Canestrelli, I. L., Meyer, T. E., Cusanovich, M. A., Getzoff, E. D., and Tollin, G. Biochemistry (1998) 37, 11563-11568]. The FTIR difference spectrum of PYP(M) and the ground state of M100L demonstrated extensive peptide-backbone structural changes as observed in the FTIR difference spectrum of the wild-type protein and PYP(M). The conformational change investigated by CD spectroscopy in the far-UV region showed reduction of the alpha-helical content by approximately 40%, indicating a considerable amount of changes in the secondary structure. The optical activity of the p-coumaric acid chromophore completely vanished upon PYP(M) in contrast to the dark state, indicating deformation of the binding pocket structure in PYP(M). The tertiary structural changes were further monitored by small-angle X-ray scattering measurements, which demonstrated a significant increase of the radius of gyration of the molecule by approximately 5% in PYP(M). These structural changes were reversed concomitantly with the chromophore anionization upon the dark state recovery. The observed changes of the quantities provided a more vivid view of the structural changes of the mutant PYP in going from PYP(M) to PYP(dark), which can be regarded as a process of folding of the secondary and the tertiary structures of the "PAS" domain structure, coupled with the p-coumaric acid chromophore deprotonation and isomerization.

Bacterial Proteins↗

PubMed2002

Structural change of site-directed mutants of PYP: new dynamics during pR state.

The energetics, protein dynamics, and diffusion coefficients of three mutants of photoactive yellow protein, R52Q, P68A, and W119G, were studied by the transient grating and pulsed laser-induced photoacoustic method. We observed a new dynamics with a lifetime of approximately 1 micro s in the transient grating signal, which is silent by the light absorption technique. This fact indicates that, after the structure change around the chromophore is completed (pR(1)), the protein part located far from the chromophore is still moving to finally create another pR (pR(2)) species, which can transform to the next intermediate, pB. Although the kinetics of pR(2)-->pB-->pG are very different depending on the mutants, the enthalpies of the first long-lived (in micro seconds, 100-micro s range) intermediate species (pR(2)) are similar and very high for all mutants. The diffusion coefficients of the parent (pG) and pB species of the mutants are also similar to that of the wild-type photoactive yellow protein. From the temperature dependence of the volume change, the difference in the thermal expansion coefficients taken as indicator of the flexibility of the structure between pG and pR(2) is measured. They are also similar to that of the wild-type photoactive yellow protein. These results suggest that the protein structures of pR(2) and pB in these mutants are globally different from that of pG, and this structural change is not altered so much by the single amino acid residue mutation. This is consistent with the partially unfolded nature of these intermediate species. On the other hand, the volume changes during pR(1)-->pR(2) are sensitive to the mutations, which may suggest that the volume change reflects a rather local character of the structure, such as the chromophore-protein interaction.

Acoustics↗

PubMed2002

Time-resolved X-ray diffraction reveals movement of F helix of D96N bacteriorhodopsin during M-MN transition at neutral pH.

D96N bacteriorhodopsin has two photointermediates with the deprotonated Schiff base: the M and MN intermediates. We measure the time-resolved x-ray diffraction of the D96N purple membrane after flash photoexcitation (pH 7.0, 25 degrees C). The data clearly show the M-MN transition during the D96N photocycle. Low-resolution projection maps of these states show that the F helix of the MN intermediate shifts from its original position and this shift is much larger than that of the M intermediate. This indicates that the F helix moves in the M-MN transition of the D96N bacteriorhodopsin photocycle. Moreover, the existence of the MN intermediate in the D96N photocycle under neutral pH indicates that the MN intermediate is not peculiar to the alkaline condition. It is notable that the structural transition of M-MN is independent of the protonation state of the Schiff base. Therefore, the F helix movement precedes reprotonation of the Schiff base in the bacteriorhodopsin photocycle. Our previous study showed that the M-MN transition is hydration-dependent and that the MN intermediate is more hydrated than the M intermediate. Considering this together with the present results, we conclude that the movement of the F helix causes hydration of the cytoplasmic side, which promotes the reprotonation of the Schiff base.

Amino Acid Substitution↗

PubMed2002

Distribution of rod- and cone-specific phosducins in retinas of non-mammalian vertebrates.

In mammalian retinas, it has been believed that just one kind of phosducin (PD) commonly exists in both rods and cones. However, we have previously reported that there are rod- and cone-specific PDs (OlPD-R and OlPD-C) in medaka (Oryzias latipes) retina [FEBS Lett., 502, 117-121, 2001]. To clarify the distribution and evolution of these photoreceptor type-specific PDs, we investigated PDs of another teleost and a reptile. Immunohistochemical and Western blot analyses using anti-medaka PD antisera demonstrated that two kinds of PDs are expressed in zebrafish (Danio rerio) photoreceptor cells. Our study is suggestive that teleosts generally possess rod- and cone-specific PDs. We isolated a cDNA encoding putative PD (PmlPD) of a diurnal gecko (Phelsuma madagascariensis longinsulae). Because diurnal gecko possesses a pure-cone retina, it was expected that PmlPD would be expressed in cones. Molecular phylogenetic analysis demonstrated that PmlPD was more closely related to mammalian PDs than teleost cone-specific PDs, suggesting that the rod- and cone-specific subtype of teleost PDs have arisen after the teleost-tetrapod divergence.

Amino Acid Sequence↗

PubMed2002

Molecular evolution of proteins involved in vertebrate phototransduction.

Vision is one of the most important senses for vertebrates. As a result, vertebrates have evolved a highly organized system of retinal photoreceptors. Light triggers an enzymatic cascade, called the phototransduction cascade, that leads to the hyperpolarization of photoreceptors. It is expected that a systematic comparison of phototransduction cascades of various vertebrates can provide insights into the diversity of vertebrate photoreceptors and into the evolution of vertebrate vision. However, only a few attempts have been made to compare each phototransduction protein participating in this cascade. Here, we determine phylogenetic trees of the vertebrate phototransduction proteins and compare them. It is demonstrated that vertebrate opsin sequences fall into five fundamental subfamilies. It is speculated that this is crucial for the diversity of the spectral sensitivity observed in vertebrate photoreceptors and provides the vertebrates with the molecular tools to discriminate the color of incident light. Other phototransduction proteins can be classified into only a few subfamilies. Cones generally share isoforms of phototransduction proteins that are different from those found in rods. The difference in sensitivity to light between rods and cones is likely due to the difference in the molecular properties of these isoforms. The phototransduction proteins seem to have co-evolved as a system. Switching the expression of these isoforms may characterize individual vertebrate photoreceptors.

Amino Acid Sequence↗