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F Siebert

Publications and source records attributed to F Siebert.

At least 37 records · Page 2Linked to original sources
PubMed1997

Z,E isomerization of the alpha-84 phycoviolobilin chromophore of phycoerythrocyanin from Mastigocladus laminosus investigated by Fourier-transform infrared difference spectroscopy.

The photoreaction of the phycoviolobilin (PVB) chromophore-containing part of phycoerythrocyanin (PEC) from Mastigocladus laminosus was investigated by Fourier-transform infrared spectroscopy (FT-IR). Difference spectra between the parent states P566 and P507 were obtained in 1H2O and 2H2O for the first time. The spectra are generally characterised by large changes in the range between 1710 and 1590 cm(-1) and by a strong difference band around 1270 cm(-1). In order to study the influence on the PVB chromophore upon aggregation, spectra of the alpha-subunit and the (alphabeta)3 trimer are compared, showing distinct differences which may be of relevance for the chromophore-protein and protein-protein interactions. The difference spectra demonstrate many similarities to the spectra recently obtained for the Pr --> meta-Rc transition of phytochrome [Foerstendorf et al. (1996) Biochemistry 35, 10793-10799]. In particular, a band around 1710 cm(-1), which was tentatively assigned to the C = O stretch of ring D is also observed in the spectra of PEC. It strongly supports this identification and the deduced molecular interpretation on the protonation state of the chromophore.

Cyanobacteria↗
PubMed1997

A first report of familial visceral myopathy with additional chylous ascites in two siblings.

BACKGROUND: Familial visceral myopathy in two siblings, involving the intestine with concurrent chylous ascites and chylous pleural effusion, is reported. In this connection a review of the recent gastroenterologic literature was performed. PATIENTS: In two siblings, admitted between 1992 and 1995 with increasing ascites, loss of weight, and attacks of subileus, the diagnosis of familial visceral myopathy was established. CASE ONE: During the initial preoperative examination hepatogenous ascites was diagnosed. Visceral myopathy and chylous ascites were subsequently established by means of an explorative laparotomy in conjunction with histologic examination of specimens taken from the small-bowel wall. CASE TWO: The patient's brother was admitted 3 years later with similar symptoms. Complaints of chylous ascites and chylous pleural effusion in connection with attacks of subileus are still present. OUTCOME: The female sibling was discharged from hospital with unchanged complaints and died only a few months later; the male sibling is still alive, but with unchanged complaints. CONCLUSIONS: Rare cases of familial and sporadic myopathy have been reported in the recent literature, but to our knowledge these are the first cases with concurrent chylous ascites and chylous pleural effusion in this uncommon disorder.

Aged↗
PubMed1996

Spectroscopic evidence for altered chromophore--protein interactions in low-temperature photoproducts of the visual pigment responsible for congenital night blindness.

The replacement of Gly90 by Asp in human rhodopsin causes congenital night blindness. It has been suggested that the molecular origin for the trait is an altered electrostatic environment of the protonated retinal Schiff base chromophore. We have investigated the corresponding recombinant bovine rhodopsin mutant G90D, as well as the related mutants E113A and G90D/E113A, using spectroscopy at low temperature. This allows the assessment of chromophore-protein interactions under conditions where conformational changes are mainly restricted to the retinal-binding site. Each of the mutant pigments formed bathorhodopsin- and isorhodopsin-like intermediates, but the concomitant visible absorption changes reflected differences in the electrostatic environment of the protonated Schiff base in each pigment. Fourier transform infrared-difference spectroscopy revealed effects on the chromophore fingerprint and hydrogen-out-of-plane vibrational modes, which were indicative of the removal of an electrostatic perturbation near C12 of the retinal chromophore in all three mutants. A comparison of the UV-visible and infrared-difference spectra of the mutant pigments strongly suggests that Glu113 is stably protonated in G90D. The corresponding carbonyl-stretching mode is assigned to a band at 1727 cm-1. In contrast to the case in native bathorhodopsin, the all-trans-retinal chromophores in the primary photoproducts of the mutant pigments are essentially relaxed. The peptide carbonyl vibrational changes in mutants G90D and G90D/ E113A suggest that this is due to a more flexible retinal-binding site. Therefore, the steric strain exerted on the chromophore in native bathorhodopsin may be caused by electrostatic forces that specifically involve glutamate 113.

Amides↗
PubMed1996

Protonation changes during the photocycle of sensory rhodopsin II from Natronobacterium pharaonis.

The Fourier Transform Infrared (FTIR) spectra of photocycle intermediates of sensory rhodopsin II (pSRII) from Natronobacterium pharaonis were measured. The results of the FTIR experiments indicate considerable conformational movements of pSRII already at the stage of the early K-like intermediate which persist at least during the lifetime of the long lived intermediate. These changes in the amide bond region are more intense than those described for sensory rhodopsin I (SRI) and are quite similar to those observed for rhodopsin. Concomitantly with the deprotonation of the Schiff base a carboxyl group located in a hydrophobic environment is protonated. In analogy to bacteriorhodopsin, this carboxyl group might arise from Asp-75 which probably serves as counter ion to the Schiff base. The protonation reaction differs from the situation observed in SRI where the protonation is pH independent over the range of pH 5-8.

Archaea↗
PubMed1996

Steric interaction between the 9-methyl group of the retinal and tryptophan 182 controls 13-cis to all-trans reisomerization and proton uptake in the bacteriorhodopsin photocycle.

The hypothesis was tested whether in bacteriorhodopsin (BR) the reduction of the steric interaction between the 9-methyl group of the chromophore all-trans-retinal and the tryptophan at position 182 causes the same changes as observed in the photocycle of 9-demethyl-BR. For this, the photocycle of the mutant W182F was investigated by time-resolved UV-vis and pH measurements and by static and time-resolved FT-IR difference spectroscopy. We found that the second half of the photocycle was similarly distorted in the two modified systems: based on the amide-I band, the protonation state of D96, and the kinetics of proton uptake, four N intermediates could be identified, the last one having a lifetime of several seconds; no O intermediate could be detected; the proton uptake showed a pronounced biphasic time course; and the pKa of group(s) on the cytoplasmic side in N was reduced from 11 in wild type BR to around 7.5. In contrast to 9-demethyl-BR, in the W182F mutant the first part of the photocycle does not drastically deviate from that of wild type BR. The results demonstrate the importance of the steric interaction between W182 and the 9-methyl group of the retinal in providing tight coupling between chromophore isomerization and the late proton transfer steps.

Halobacterium↗
PubMed1996

Fourier-transform infrared spectroscopy of phytochrome: difference spectra of the intermediates of the photoreactions.

The photocycle of 124 kDa phytochrome A from Avena sativa was studied by Fourier-transform infrared spectroscopy at low temperatures. Difference spectra between the parent state Pr and the intermediates of the Pr-->Pfr pathway, i.e. lumi-R, meta-Ra, and meta-Rc, and between Pfr and the intermediates of the Pfr-->Pr pathway, lumi-F and meta-F, were obtained in 1H2O and 2H2O for the first time. Each spectrum shows characteristic spectral features which allow a clear distinction between the different intermediates. A general feature is that greater changes occur with increasing temperature, i.e. at the later steps of the photoreactions. Nevertheless, the changes in the spectral regions of the protein (amide I and amide II) were found to be surprisingly small, excluding larger conformational changes of the protein. All spectra of the intermediates are characterized by a strong negative band around 1700 cm-1. This band is tentatively assigned to the C = O stretch of ring D of the chromophore. Since it is not observed in the difference spectra between the parent states, it is concluded that ring D is located in a similar molecular environment in Pr and Pfr. In the photoproducts lumi-R and lumi-F, this band undergoes an upshift to 1720 cm-1. The high frequencies suggest that the chromophore is protonated in these intermediates as well as in Pr and Pfr.

Avena↗
PubMed1996

Characterization of the mutant visual pigment responsible for congenital night blindness: a biochemical and Fourier-transform infrared spectroscopy study.

A mutation in the gene for the rod photoreceptor molecule rhodopsin causes congenital night blindness. The mutation results in a replacement of Gly90 by an aspartic acid residue. Two molecular mechanisms have been proposed to explain the physiology of affected rod cells. One involves constitutive activity of the G90D mutant opsin [Rao, V. R., Cohen, G. B., & Oprian, D. D. (1994) Nature 367, 639-642]. A second involves increased photoreceptor noise caused by thermal isomerization of the G90D pigment chromophore [Sieving, P. A., Richards, J. E., Naarendorp F., Bingham, E. L., Scott, K., & Alpern, M. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 880-884]. Based on existing models of rhodopsin and in vitro biochemical studies of site-directed mutants, it appears likely that Gly90 is in the immediate proximity of the Schiff base chromophore linkage. We have studied in detail the mutant pigments G90D and G90D/E113A using biochemical and Fourier-transform infrared (FTIR) spectroscopic methods. The photoproduct of mutant pigment G90D, which absorbs maximally at 468 nm and contains a protonated Schiff base linkage, can activate transducin. However, the active photoproduct decays rapidly to opsin and free all-trans-retinal. FTIR studies of mutant G90D show that the dark state of the pigment has several structural features of metarhodopsin II, the active form of rhodopsin. These include a protonated carboxylic acid group at position Glu113 and increased hydrogen-bond strength of Asp83. Additional results, which relate to the structure of the active G90D photoproduct, are also reported. Taken together, these results may be relevant to understanding the molecular mechanism of congenital night blindness caused by the G90D mutation in human rhodopsin.

Aspartic Acid↗
PubMed1996

Solid-state 13C-NMR of [(3-13C)Pro]bacteriorhodopsin and [(4-13C)Pro]bacteriorhodopsin: evidence for a flexible segment of the C-terminal tail.

The configuration of an Xaa-Pro bond can be determined by solid-state magic-angle-sample-spinning (MASS)-13C-NMR spectroscopy since the chemical shifts of C beta and Cgamma of the proline ring are sensitive to the isomerization state of the preceding peptide bond. (3-13C)Pro and (4-13C)Pro have been chemically synthesized; the former by means of an asymmetric synthesis. The 13C-labeled Pro residues were biosynthetically incorporated into bacteriorhodopsin with a yield of 80%. The solid-state-MASS-13C-NMR spectra of [(3-13C)Pro]bacteriorhodopsin and [(4-13C)Pro]bacteriorhodopsin revealed isotropic chemical shifts at 29.8 ppm and 25.5 ppm, respectively. From the chemical-shift values we conclude that all Xaa Pro peptide bonds are in the trans configuration confirming previous results from solution-NMR studies on solubilized bacteriorhodopsin in organic solvents [Deber, M.C., Sorrell, B.J. & Xu, G.Y. (1990) Biochem. Biophys. Res. Commun. 172, 862-869]. Inversion-recovery experiments could differentiate between three classes of Pro residues distinguished by their relaxation time t1. Tentatively, these three distinct groups of Pro residues could be assigned to the helical, the loop, and the C-terminal parts of the protein. The resonances of the two C-terminal Pro could be identified by removing the C-terminus by proteolysis. Although they are separated by only one Glu they occupy different chemical environments and possess different flexibilities. These results indicate that the first part of the C-terminal tail is constrained. Pro238 marks the position where the tail becomes freely mobile. It is proposed that the C-terminus is fixed to the membrane via salt bridges between divalent cations and negative charges of the C-terminus as well as interhelical loops.

Bacteriorhodopsins↗
PubMed1995

Influence of the 9-methyl group of the retinal on the photocycle of bacteriorhodopsin studied by time-resolved rapid-scan and static low-temperature Fourier transform infrared difference spectroscopy.

The photocycle of bacteriorhodopsin (BR) regenerated with all-trans-9-demethylretinal was investigated by time-resolved rapid-scan Fourier transform infrared difference spectroscopy, by static low-temperature difference spectroscopy at 80, 170, and 213 K and by static steady-state difference spectroscopy at 278 K. In addition, the formation and decay of M intermediate was monitored at 412 nm with conventional flash photolysis experiments. Our data show that the removal of the 9-methyl group strongly changes the photocycle of BR. The reaction cycle is slowed down about 250-fold. The photoreaction is characterized by a slow rise of the M intermediate and by a very long-lived N intermediate. No O intermediate could be observed. The low-temperature spectra indicate that already at 80 K a KL-like photoproduct is formed. L can be obtained as in native BR at 170 K, but its decay appears to be inhibited, since it can still be observed at 213 K and high pH, in addition to the M intermediate. As in native BR, the 15-hydrogen out-of-plane modes of the L and N intermediates (observed in 2H2O) are very similar. Evidence for the existence of three N substates which differ in the protonation state of Asp96 and in the amide I bands is presented. This is explained by the extremely slowed-down reisomerization of the chromophore. The results are discussed with respect to alterations in the chromophore-protein interaction, caused by the removal of the 9-methyl group.

Bacteriorhodopsins↗
PubMed1994

A mutant rhodopsin photoproduct with a protonated Schiff base displays an active-state conformation: a Fourier-transform infrared spectroscopy study.

In the rhodopsin mutant E113A/A117E the position of the protonated Schiff base counterion, Glu113, is moved by one helix turn from position 113 to 117. The photoreaction of this mutant pigment was studied by Fourier-transform infrared (FTIR) difference spectroscopy. At acidic pH, formation of a 474-nm absorbing photoproduct previously characterized biochemically as a species that activates transducin caused infrared absorption changes typical of metarhodopsin II (MII) formation in native rhodopsin. Specific spectral alterations revealed a localized perturbation near the protonated Schiff base in the dark state. In addition, an infrared band assigned to the C = O stretching vibration of Glu113 in MII of rhodopsin was abolished in the mutant. Absorption changes caused by Asp83 and Glu122 C = O stretching vibrations characteristic of rhodopsin MII formation were not affected. At alkaline pH, mutant E113A/A117E formed predominantly a 382-nm absorbing photoproduct. It displayed infrared-difference absorption bands significantly different from those of native MII over a large spectral range. These results support the conclusion that the 474-nm photoproduct of mutant E113A/A117E, despite a protonated Schiff base linkage, displays a predominantly MII-like conformation capable of catalyzing guanine-nucleotide exchange by transducin.

Carboxylic Acids↗
PubMed1994

Structural changes of sarcoplasmic reticulum Ca(2+)-ATPase upon Ca2+ binding studied by simultaneous measurement of infrared absorbance changes and changes of intrinsic protein fluorescence.

Ca2+ binding to sarcoplasmic reticulum Ca(2+)-ATPase was investigated by Fourier transform infrared (FTIR) spectroscopy using the photolytic release of Ca2+ from the photolabile Ca2+ chelator 1-(2-nitro-4,5-dimethoxy)-N,N,N',N',- tetrakis[(oxycarbonyl)]methyl-1,2-ethandiamine (DM-nitrophen). IR absorbance changes in 1H2O and 2H2O were detected in the spectral region from 1800 cm-1 to 1200 cm-1, reflecting photolysis of DM-nitrophen and Ca2+ binding to the Ca(2+)-ATPase. As an independent probe for protein conformational changes, intrinsic fluorescence changes upon Ca2+ release were monitored simultaneously to the FTIR measurements. Both the IR absorbance changes and the fluorescence intensity changes correlated well with the Ca2+ binding activity of the ATPase in this specific step. Ca2+ binding caused IR difference bands mainly in the region of amide I absorption of the polypeptide backbone, reflecting conformational changes of the protein. The small amplitude of the signals indicates that only a few residues perform local structural changes such as changes of bond angles or hydrogen bonding. Other absorbance changes appearing above 1700 cm-1 can be assigned to Ca2+ binding to Glu or Asp side chain carboxyl groups and concomitant deprotonation of these residues. This assignment is strengthened by downshifts of these bands by 4 cm-1 to 6 cm-1 upon 1H2O/2H2O exchange. This is in line with results of mutagenesis studies where such residues containing carboxyl groups were associated with the high affinity Ca2+ binding site (Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1990) J. Biol. Chem. 265, 6262-6267).

Animals↗
PubMed1994

Identification of glutamic acid 113 as the Schiff base proton acceptor in the metarhodopsin II photointermediate of rhodopsin.

In order to investigate the molecular mechanism of rhodopsin photoactivation, site-directed mutants of bovine rhodopsin were studied by Fourier-transform infrared (FTIR) difference spectroscopy. Rhodopsin mutants E113D and E113A were prepared in which the retinylidene Schiff base counterion, Glu113, was replaced by Asp and Ala, respectively. FTIR difference spectra were recorded and compared with spectra of recombinant native rhodopsin. Both mutant pigments formed photoproducts at 0 degrees C with vibrational absorption bands typical of the metarhodopsin II (MII) state of rhodopsin. The FTIR difference spectrum of E113D was nearly identical to that of rhodopsin. A positive band at 1712 cm-1 caused by the protonation of an internal carboxylic acid in rhodopsin was shifted slightly to 1709 cm-1 in mutant E113D. E113A was studied at acidic pH in the presence of chloride as an inorganic counterion to the protonated Schiff base. The 1712-cm-1 (1709-cm-1) band was absent in the FTIR difference spectrum of mutant E113A. Therefore, we have assigned the 1712-cm-1 absorbance band to the C = O stretching vibration of protonated Glu113 in MII of rhodopsin. These results show that the Schiff base counterion of rhodopsin, the carboxylate side chain of Glu113, becomes protonated during MII formation.

Animals↗
PubMed1994

Interactions of the beta-ionone ring with the protein in the visual pigment rhodopsin control the activation mechanism. An FTIR and fluorescence study on artificial vertebrate rhodopsins.

The photoreactions of rhodopsin regenerated with three 9-cis retinal analogs, modified at or in the vicinity of the beta-ionone ring (namely 5,6-epoxy, 7,8-diH, diethyl-acyclic) have been investigated by UV-vis and FTIR difference spectroscopy. In parallel, the ability to catalyze the GDP-->GTP exchange of G-protein (transducin) has been monitored by time-dependent fluorescence spectroscopy. The first photoproduct obtained with all three pigments at liquid nitrogen temperature is a blue-shifted intermediate (BSI), followed by a lumi-like intermediate at 170 K. For the 5,6-epoxy-ISO and 7,8-diH-ISO pigment we obtain two further intermediates similar to the META-I and META-II states of native RHO. For the diethyl-acyclic-ISO pigment only one further intermediate can be stabilized at 280 K. As compared to META-II the respective photoproduct exhibits striking differences. The latter two pigments have also been investigated in the solubilized lipid-free state (detergent: dodecyl maltoside) at 280 K. For the 5,6-epoxy-ISO pigment, the UV-vis, FTIR, and activation data agree with the formation of a META-II-like photoproduct (81% activation). Less META-II formation is observed for the 7,8-dihydro-ISO pigment in membranes (65% activation), but full formation in detergent (100% activation). Neither the membrane-bound nor the solubilized diethyl-acyclic-ISO pigment forms a META-II-like intermediate (18% and 0% activation, respectively). Therefore, we conclude that the substitution of the beta-ionone ring by two ethyl groups abolishes steric interactions with the protein, which are essential for META-II formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Protonation states of membrane-embedded carboxylic acid groups in rhodopsin and metarhodopsin II: a Fourier-transform infrared spectroscopy study of site-directed mutants.

A method was developed to measure Fourier-transform infrared (FTIR) difference spectra of detergent-solubilized rhodopsin expressed in COS cells. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and three expressed rhodopsin mutants with amino acid replacements of membrane-embedded carboxylic acid groups: Asp-83-->Asn (D83N), Glu-122-->Gln (E122Q), and the double mutant D83N/E122Q. Each of the mutant opsins bound 11-cis-retinal to yield a visible light-absorbing pigment. Upon illumination, each of the mutant pigments formed a metarhodopsin II-like species with maximal absorption at 380 nm that was able to activate guanine nucleotide exchange by transducin. Rhodopsin versus metarhodopsin II-like photoproduct FTIR-difference spectra were recorded for each sample. The COS-cell rhodopsin and mutant difference spectra showed close correspondence to that of rhodopsin from disc membranes. Difference bands (rhodopsin/metarhodopsin II) at 1767/1750 cm-1 and at 1734/1745 cm-1 were absent from the spectra of mutants D83N and E122Q, respectively. Both bands were absent from the spectrum of the double mutant D83N/E122Q. These results show that Asp-83 and Glu-122 are protonated both in rhodopsin and in metarhodopsin II, in agreement with the isotope effects observed in spectra measured in 2H2O. A photoproduct band at 1712 cm-1 was not affected by either single or double replacements at positions 83 and 122. We deduce that the 1712 cm-1 band arises from the protonation of Glu-113 in metarhodopsin II.

Amino Acid Sequence↗
PubMed1993

Aspartic acid-212 of bacteriorhodopsin is ionized in the M and N photocycle intermediates: an FTIR study on specifically 13C-labeled reconstituted purple membranes.

Purple membrane was regenerated from the denatured proteolytic (protease V8) fragments V-1 and V-2 of bacteriorhodopsin (BR), native membrane lipids, and all-trans-retinal. FTIR difference spectra of M and N intermediates of the reconstituted system are in close correspondence to those obtained from native BR. Asp-212 is the only internal aspartic acid in the V-2 fragment (helices F and G). Reconstituting a V-2 fragment from a [4-13C]Asp-labeled BR preparation with an unmodified V-1 fragment and vice versa have allowed us to assign IR bands to either Asp-212 or any of the remaining aspartic acids on V-1 (helices A-E). A carboxylate vibration at 1392 cm-1 has been identified in the M and N intermediates and assigned to Asp-212. Since no contribution of this residue to C = O stretches of protonated carboxyl groups was detected, Asp-212 must be ionized in light-adapted BR as well. The effect of [4-13C]Asp labeling of V-1 revealed a carboxylate vibration at 1385 cm-1 in light-adapted BR. Since Asp-96 and Asp-115 are protonated, this band is caused by Asp-85. All absorption changes of C = O stretches of protonated carboxyl groups are due to Asp residues on V-1. Correspondingly, the proton acceptor for Schiff base deprotonation in M is located on V-1, and must be Asp-85 (the only ionized Asp on V-1). The band assignments are compared with those reported for BR mutants, and the potential role of Asp-212 for proton translocation is discussed.

Aspartic Acid↗
PubMed1992

Asp85 is the only internal aspartic acid that gets protonated in the M intermediate and the purple-to-blue transition of bacteriorhodopsin. A solid-state 13C CP-MAS NMR investigation.

High-resolution solid-state 13C NMR spectra of the ground state and M intermediate of the bacteriorhodopsin mutant D96N with the isotope label at [4-13C]Asp and [11-13C]Trp were recorded. The NMR spectra show that Asp85 is protonated in the M intermediate. The environment of Asp85 is quite hydrophobic. On the other hand, Asp212 remains deprotonated and a slight shift to lower field indicates a more hydrophilic environment. Asp85 also protonates in the purple-to-blue transition of bacteriorhodopsin in the deionized membrane, where it experiences a similar environment to M. The shift of Trp resonances in M reflect a conformational change of the protein in forming the M intermediate.

Aspartic Acid↗
PubMed1992

High-resolution solid state 13C NMR of bacteriorhodopsin: characterization of [4-13C]Asp resonances.

Solid state 13C nuclear magnetic resonance measurements of bacteriorhodopsin labeled with [4-13C]Asp show that resonances of single amino acids can be resolved. In order to assign and characterize the resonances of specific Asp residues, three different approaches were used. (1) Determination of the chemical shift anisotropy from side-band intensities provides information about the protonation state of Asp residues. (2) Relaxation studies and T1 filtering allow one to discriminate between resonances with different mobility. (3) A comparison of the spectra of light- and dark-adapted bacteriorhodopsin provides evidence for resonances from aspartic acid residues in close neighborhood of the chromophore. In agreement with other investigations, four resonances are assigned to internal residues. Two of them are protonated in the ground state up to pH 10 (Asp96 and Asp115). All other detected resonances, including Asp85 and Asp212, are due to deprotonated aspartic acid. Two lines due to the two internal deprotonated groups change upon dark and light adaptation, whereas the protonated Asp residues are unaffected.

Amino Acid Sequence↗