Biomedical subjects

W D Hoff

Publications and source records attributed to W D Hoff.

28 records · Page 2Linked to original sources

PubMed1995

Resonance Raman evidence that the thioester-linked 4-hydroxycinnamyl chromophore of photoactive yellow protein is deprotonated.

Resonance Raman spectra of the ground state of photoactive yellow protein (PYP), a photoactive pigment found in Ectothiorhodospira halophila, have been obtained with excitation at 413.1 nm using a microspinning sample cell. The resonance Raman spectra of the thioester-linked 4-hydroxycinnamyl chromophore in the protein are compared with the preresonance Raman spectra of the 4-hydroxycinnamyl phenyl thioester and 4-hydroxycinnamic acid model compounds at various pH values. Bands at 1568, 1542, 1500, 1434, and 1166 cm-1 in the Raman spectrum of the anionic form of the 4-hydroxycinnamyl phenyl thioester are shown to be characteristic for the deprotonation of the chromophore. The observation of bands in PYP exhibiting very similar frequency and intensity patterns provides strong evidence that the chromophore in PYP is stabilized as a phenolate anion at pH 7.4, in support of conclusions from crystallographic studies. Furthermore, the insensitivity of the PYP Raman spectrum to placement of the protein in D2O buffer is consistent with the absence of the exchangeable phenolic proton on the cinnamyl chromophore. These results establish the feasibility of elucidating the molecular mechanism of light-to-information transduction by this new photosensory pigment with resonance Raman spectroscopy.

Bacterial Proteins↗

PubMed1995

Photoinduced volume change and energy storage associated with the early transformations of the photoactive yellow protein from Ectothiorhodospira halophila.

The photocycle of the photoactive yellow protein (PYP) isolated from Ectothiorhodospira halophila was analyzed by flash photolysis with absorption detection at low excitation photon densities and by temperature-dependent laser-induced optoacoustic spectroscopy (LIOAS). The quantum yield for the bleaching recovery of PYP, assumed to be identical to that for the phototransformation of PYP (pG), to the red-shifted intermediate, pR, was phi R = 0.35 +/- 0.05, much lower than the value of 0.64 reported in the literature. With this value and the LIOAS data, an energy content for pR of 120 kJ/mol was obtained, approximately 50% lower than for excited pG. Concomitant with the photochemical process, a volume contraction of 14 ml/photoconverted mol was observed, comparable with the contraction (11 ml/mol) determined for the bacteriorhodopsin monomer. The contraction in both cases is interpreted to arise from a protein reorganization around a phototransformed chromophore with a dipole moment different from that of the initial state. The deviations from linearity of the LIOAS data at photon densities > 0.3 photons per molecule are explained by absorption by pG and pR during the laser pulse duration (i.e., a four-level system, pG, pR, and their respective excited states). The data can be fitted either by a simple saturation process or by a photochromic equilibrium between pG and pR, similar to that established between the parent chromoprotein and the first intermediate(s) in other biological photoreceptors. This nonlinearity has important consequences for the interpretation of the data obtained from in vitro studies with powerful lasers.

Bacterial Proteins↗

PubMed1994

Thiol ester-linked p-coumaric acid as a new photoactive prosthetic group in a protein with rhodopsin-like photochemistry.

A number of Eubacteria contain a photoactive yellow protein which has a photosensory function in negative phototaxis. It has been proposed that the cofactor responsible for the intense yellow color of this protein is retinal [McRee, D. E., et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6533-6537]. This would make it the first eubacterial rhodopsin. Here we report the chemical structure of this chromophoric group to be p-coumaric acid, which is covalently bound to a unique cysteine in the apoprotein via a thiol ester bond, and thus not retinal. This makes PYP the first example of a protein containing p-coumaric acid, a metabolite previously found only in plants, as a prosthetic group and establishes the photoactive yellow proteins as a new type of photochemically active receptor molecule.

Chromatiaceae↗

PubMed1994

Photobiology of bacteria.

The field of photobiology is concerned with the interactions between light and living matter. For Bacteria this interaction serves three recognisable physiological functions: provision of energy, protection against excess radiation and signalling (for motility and gene expression). The chemical structure of the primary light-absorbing components in biology (the chromophores of photoactive proteins) is surprisingly simple: tetrapyrroles, polyenes and derivatised aromats are the most abundant ones. The same is true for the photochemistry that is catalysed by these chromophores: this is limited to light-induced exciton- or electron-transfer and photoisomerization. The apoproteins surrounding the chromophores provide them with the required specificity to function in various aspects of photosynthesis, photorepair, photoprotection and photosignalling. Particularly in photosynthesis several of these processes have been resolved in great detail, for others at best only a physiological description can be given. In this contribution we discuss selected examples from various parts of the field of photobiology of Bacteria. Most examples have been taken from the purple bacteria and the cyanobacteria, with special emphasis on recently characterised signalling photoreceptors in Ectothiorhodospira halophila and in Fremyella diplosiphon.

Bacteria↗

PubMed1994

Measurement and global analysis of the absorbance changes in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila.

The photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila was examined by time-resolved difference absorption spectroscopy in the wavelength range of 300-600 nm. Both time-gated spectra and single wavelength traces were measured. Global analysis of the data established that in the time domain between 5 ns and 2 s only two intermediates are involved in the room temperature photocycle of PYP, as has been proposed before (Meyer T.E., E. Yakali, M. A. Cusanovich, and G. Tollin. 1987. Biochemistry. 26:418-423; Meyer, T. E., G. Tollin, T. P. Causgrove, P. Cheng, and R. E. Blankenship. 1991. Biophys. J. 59:988-991). The first, red-shifted intermediate decays biexponentially (60% with tau = 0.25 ms and 40% with tau = 1.2 ms) to a blue-shifted intermediate. The last step of the photocycle is the biexponential (93% with tau = 0.15 s and 7% with tau = 2.0 s) recovery to the ground state of the protein. Reconstruction of the absolute spectra of these photointermediates yielded absorbance maxima of about 465 and 355 nm for the red- and blue-shifted intermediate with an epsilon max at about 50% and 40% relative to the epsilon max of the ground state. The quantitative analysis of the photocycle in PYP described here paves the way to a detailed biophysical analysis of the processes occurring in this photoreceptor molecule.

Bacterial Proteins↗

PubMed1994

The photoactive yellow protein from Ectothiorhodospira halophila as studied with a highly specific polyclonal antiserum: (intra)cellular localization, regulation of expression, and taxonomic distribution of cross-reacting proteins.

A rabbit antiserum was raised against the photoactive yellow protein (PYP) from Ectothiorhodospira halophila and purified by adsorption experiments to obtain a highly specific polyclonal antiserum. This antiserum was used to obtain the following results. (i) In E. halophila, PYP can be isolated from the fraction of soluble proteins. In the intact cell, however, PYP appeared to be associated with (intra)cytoplasmic membranes, as was concluded from analysis of immunogold-labelled thin sections of the organism. (ii) The regulation of expression of PYP was studied by using dot blot assays, Western blotting (immunoblotting), and rocket immunoelectrophoresis. Under all conditions investigated (light color, salt concentration, and growth phase), PYP was expressed constitutively in E. halophila. However, when Rhodospirillum salexigens was grown aerobically, the expression of PYP was suppressed. (iii) A large number of prokaryotic microorganisms contained a single protein, with an apparent size of approximately 15 kDa, that cross-reacted with the antiserum. Among the positively reacting organisms were both phototrophic and chemotrophic, as well as motile and nonmotile, organisms. After separation of cellular proteins into a membrane fraction and soluble proteins, it was established that organisms adapted to growth at higher salt concentrations tended to have the cross-reacting protein in the soluble fraction. In the cases of R. salexigens and Chromatium salexigens, we have shown that the cross-reacting protein involved is strongly homologous to PYP from E. halophila.

Amino Acid Sequence↗

PubMed1993

Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila, with evidence for the mass and the binding site of the chromophore.

The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP+ ++ CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4 A resolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+/- 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.

Amino Acid Sequence↗

PubMed1993

The eubacterium Ectothiorhodospira halophila is negatively phototactic, with a wavelength dependence that fits the absorption spectrum of the photoactive yellow protein.

The motile, alkalophilic, and extremely halophilic purple sulfur bacterium Ectothiorhodospira halophila is positively photophobotactic. This response results in the accumulation of bacteria in light spots (E. Hustede, M. Liebergesell, and H. G. Schlegel, Photochem. Photobiol. 50:809-815, 1989; D. E. McRee, J. A. Tainer, T. E. Meyer, J. Van Beeumen, M. A. Cusanovich, and E. D. Getzoff, Proc. Natl. Acad. Sci. USA 86:6533-6537, 1989; also, this work). In this study, we demonstrated that E. halophila is also negatively phototactic. Video analysis of free-swimming bacteria and the formation of cell distribution patterns as a result of light-color boundaries in an anaerobic suspension of cells revealed the existence of a repellent response toward intense (but nondamaging) blue light. In the presence of saturating background photosynthetic light, an increase in the intensity of blue light induced directional switches, whereas a decrease in intense blue light gave rise to suppression of these reversals. To our knowledge, this is the first report of a true repellent response to light in a free-swimming eubacterium, since the blue light response in Escherichia coli and Salmonella typhimurium (B. L. Taylor and D. E. Koshland, Jr., J. Bacteriol. 123:557-569, 1975), which requires an extremely high light intensity, is unlikely to be a sensory process. The wavelength dependence of this negative photoresponse was determined with narrow band pass interference filters. It showed similarity to the absorption spectrum of the photoactive yellow protein from E. halophila.

Bacteria↗

PubMed1992

Induction of nuclear lamins A/C during in vitro-induced differentiation of F9 and P19 embryonal carcinoma cells.

Lamin B is the major constituent of the nuclear lamina of undifferentiated mouse embryonal carcinoma cells. The full complement of the three major lamins A, B, and C, found in somatic mammalian cells, is acquired after induction of differentiation in vitro by certain drugs. In this study we have examined the time course of lamin A/C expression in the two embryonal carcinoma cell lines F9 and P19. We show here that lamins A/C are detectable in these cell lines, at the mRNA level and at the protein level, after 3 days of growth in media containing retinoic acid or retinoic acid + 3-isobutyl-1-methylxanthine. The data reported here indicate that the expression of lamins A/C is mainly regulated at the transcriptional level and occurs when the cells, by morphological and functional criteria, have differentiated along their developmental pathway.

1-Methyl-3-isobutylxanthine↗

PubMedDate not supplied

Rhodopsin(s) in eubacteria.

While the biochemical basis of photosynthesis by bacteriochlorophyll-based reaction centres in purple phototrophic Eubacteria and retinal-based bacteriorhodopsin in the Archaebacterium Halobacterium salinarium has been elucidated in great detail, much less is known about photosensory signal transduction; this is especially the case for Eubacteria. Recent findings on two different photosensory proteins in two different Eubacteria, which both show clear resemblances to the rhodopsins, will be presented. The photoactive yellow protein (PYP) from the purple phototrophic organism Ectothiorhodospira halophila probably functions as the photoreceptor for a new type of negative phototaxis response and has been studied in some detail with respect to its structural and photochemical characteristics. On basis of crystallographic an photochemical data it has been proposed that PYP contains retinal as a chromophore. However, we have unambiguously demonstrated that the PYP chromophore is different from retinal, in spite of the fact that PYP's photochemical properties show striking similarities with the rhodopsins. The cyanobacterium Calothrix sp. displays complementary chromatic adaptation, a process in which the pigment composition of the phycobilisomes is adjusted to the spectral characteristics of the incident light. In orange light the blueish chromophore phycocyanin is present, in green light the reddish phycoerythrin is synthesized. On the basis of the action spectrum of this adaptation process, we hypothesized that a rhodopsin is the photosensor in this process. In line with this, we found that nicotine, an inhibitor of the biosynthesis of beta-carotene (which is the precursor of retinal), abolishes chromatic adaptation. Direct proof of the involvement of a photosensory rhodopsin was obtained in experiments in which the chromatic adaptation response was restored by the addition of retinal to the cultures. The two photosensory proteins mentioned above represent the first examples of eubacterial photoreceptors that can be studied at a molecular level. Our current knowledge on these two proteins and their status as retinal proteins will be reviewed.

Journal Article↗