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Biomedical subjects

F Siebert

Publications and source records attributed to F Siebert.

At least 55 records · Page 3Linked to original sources
PubMed1992

Conformational changes of cytosolic loops of bovine rhodopsin during the transition to metarhodopsin-II: an investigation by Fourier transform infrared difference spectroscopy.

In order to assign the structural changes of the protein, observed in the Fourier transform infrared (FT-IR) difference spectra of the rhodopsin-metarhodopsin-II transition, to specific regions of the protein, rhodopsin was treated by proteases. Nonilluminated and bleached rhodopsin was treated with protease K and papain. Rhodopsin digested in the bleached state was subsequently regenerated with 11-cis-retinal. From these modified samples the rhodopsin-metarhodopsin-II FT-IR difference spectra were measured. Comparing the difference spectra with that of unmodified rhodopsin, clear deviations in the amide-I and amide-II spectral range are observed. This indicates that in the unmodified pigment conformational changes of those parts of the cytosolic surface take place which are susceptible to the proteases. From the larger spectral changes obtained with samples digested in the bleached state it is concluded that the extent of modification is larger. The difference spectra of rhodopsin modified with 10 mM dithiothreitol support the existence of the 4th loop which also undergoes conformational changes. The spectral changes are interpreted in terms of a transition of an ordered structure of the loops in rhodopsin to a more random structure in metarhodopsin-II. The results demonstrate that by combining FT-IR spectroscopy with protein modification by specific proteases, conformational changes of the protein can be localized to specific regions.

Animals↗
PubMed1991

Fourier transform infrared studies of active-site-methylated rhodopsin. Implications for chromophore-protein interaction, transducin activation, and the reaction pathway.

Fourier transform infrared studies of active-site-methylated rhodopsin (ASMR) show that, as compared to unmodified rhodopsin, the photoreaction is almost unchanged up to the formation of lumirhodopsin. Especially, the deviations are much smaller than those observed for the corresponding intermediates of 13-desmethyl-rhodopsin. In metarhodopsin-I, larger alterations are present with respect to the three internal carboxyl groups. Similar deviations have been observed in meta-I of 13-desmethyl-rhodopsin. This indicates that, in agreement with our previous investigations, these carboxyl groups are located in close proximity to the chromophore. Because this latter pigment is capable, when bleached, of activating transducin, our data provide support for the earlier conclusion that deprotonation of the Schiff base is a prerequisite for transducin activation. The positions of the C = C and C - C stretching modes of the retinal suggest that the redshift observed in ASMR and its photoproducts can be explained by an increased distance of the Schiff base from the counterion(s). It is further shown that the photoreaction does not stop at metarhodopsin-I, but that this intermediate directly decays to a metarhodopsin-III-like species.

Binding Sites↗
PubMed1990

Infrared spectroscopy of phytochrome and model pigments.

Fourier-transform infrared difference spectra between the red-absorbing and far-red-absorbing forms of oat phytochrome have been measured in H2O and 2H2O. The difference spectra are compared with infrared spectra of model compounds, i.e. the (5Z,10Z,15Z)- and (5Z,10Z,15E)-isomers of 2,3,7,8,12,13,17,18-octaethyl-bilindion (Et8-bilindion), 2,3-dihydro-2,3,7,8,12,13,17,18-octaethyl-bilindion (H2Et8-bilindion), and protonated H2Et8-bilindion in various solvents. The spectra of the model compounds show that only for the protonated forms can clear differences between the two isomers be detected. Since considerable differences are present between the spectra of Et8-bilindion and H2Et8-bilindion, it is concluded that only the latter compound can serve as a model system of phytochrome. The 2H2O effect on the difference spectrum of phytochrome supports the view that the chromophore in red-absorbing phytochrome is protonated and suggests, in addition, that it is also protonated in far-red-absorbing phytochrome. The spectra show that protonated carboxyl groups are influenced. The small amplitudes in the difference spectra exclude major changes of protein secondary structure.

Fourier Analysis↗
PubMed1990

The photoreaction of active-site-methylated bacteriorhodopsin: an investigation using static and time-resolved infrared difference spectroscopy.

The photoreaction of active-site-methylated, permethylated bacteriorhodopsin has been investigated by static and time-resolved UV-vis and infrared difference spectroscopy. Additional information on the isomeric composition of the initial state and of photoproducts was obtained by retinal extraction and subsequent HPLC analysis. The data show that the dark-adapted state contains only all-trans-retinal. Prolonged illumination produces a metastable state which contains essentially only 9-cis-retinal and which decays back to the dark-adapted initial state within 8 h. The time-resolved infrared difference spectra clearly demonstrate that laser flash excitation produces an intermediate that has all the characteristics of the L intermediate. It is demonstrated that the methyl group at the Schiff base nitrogen introduces a steric hindrance with the protein which inhibits a photoreaction at 80 K, but which allows the generation of an L-like intermediate at room temperature and 173 K.

Bacteriorhodopsins↗
PubMed1990

High resolution 13C-solid state NMR of bacteriorhodopsin: assignment of specific aspartic acids and structural implications of single site mutations.

Three mutant strains of Halobacterium sp. GRB with the site of mutation in the bacterioopsin gene (PM 326: Asp96----Asn; PM 374: Asp96----Gly; PM 384: Asp85----Glu) were grown in a synthetic medium containing (4-13C)-Asp. The mutant bacteriorhodopsins labeled with (4-13C)-Asp (37%-45%), and owing to the metabolism of Halobacteria also with (11-13C)-Trp (50%-100%), were isolated as purple membranes and 13C Solid State Magic Angle Sample Spinning (MASS) Nuclear Magnetic Resonance (NMR) spectra of the samples were taken. The Asp96 mutants lacked the signal at 171.3 ppm which was previously assigned to a protonated internal Asp (Engelhard et al. 1989a). This observation supports the conclusion that Asp96 is protonated in the ground state. PM 384 (Asp85----Glu) has an absorption maximum at 610 nm. It can be converted into a purple form (lambda max = 540 nm) by treatment with a detergent (CHAPSO). The NMR-spectra of these two species differ from each other and from the wild type. The intensity of the resonance at 173 ppm in the wild type spectrum is reduced in both forms of the mutant protein. It is probable that this signal is caused by Asp85. The amino acid changes result not only in a perturbation of their direct environment but also effects on Trp residues and the chromophore protein interaction can be observed.

Aspartic Acid↗
PubMed1990

Fourier-transform Raman spectroscopy applied to photobiological systems.

Fluorescence and initiation of photoreactions are problems frequently encountered with resonance Raman spectroscopy of photobiological systems. These problems can be circumvented with Fourier-transform Raman spectroscopy by using the 1064-nm wavelength of a continuous wave neodymium-yttrium/aluminum-garnet laser as the probing beam. This wavelength is far from the absorption band of most pigments. Yet, the spectra of the investigated systems--bacteriorhodopsin, rhodopsin, and phycocyanin--show that these systems are still dominated by the chromophore, or that preresonant Raman scattering is still prevalent. Only for rhodopsin were contributions of the protein and the membrane discernible. The spectra of phycocyanin differ considerably from those obtained by excitation into the UV-absorption band. The results show the usefulness of this method and its wide applicability. In addition, analysis of the relative preresonant scattering cross sections may provide a detailed insight into the scattering mechanism.

Journal Article↗
PubMed1989

Removal of the 9-methyl group of retinal inhibits signal transduction in the visual process. A Fourier transform infrared and biochemical investigation.

The photoreaction of opsin regenerated with 9-demethylretinal has been investigated by UV-vis spectroscopy, flash photolysis experiments, and Fourier transform infrared difference spectroscopy. In addition, the capability of the illuminated pigment to activate the retinal G-protein has been tested. The photoproduct, which can be stabilized at 77 K, resembles more the lumirhodopsin species, and only minor further changes occur upon warming the sample to 170 K (stabilizing lumirhodopsin). UV-vis spectroscopy reveals no further changes at 240 K (stabilizing metarhodopsin I), but infrared difference spectroscopy shows that the protein as well as the chromophore undergoes further molecular changes which are, however, different from those observed for unmodified metarhodopsin I. UV-vis spectroscopy, flash photolysis experiments, and infrared difference spectroscopy demonstrate that an intermediate different from metarhodopsin II is produced at room temperature, of which the Schiff base is still protonated. The illuminated pigment was able to activate G-protein, as assayed by monitoring the exchange of GDP for GTP gamma S in purified G-protein, only to a very limited extent (approximately 8% as compared to rhodopsin). The results are interpreted in terms of a specific steric interaction of the 9-methyl group of the retinal in rhodopsin with the protein, which is required to initiate the molecular changes necessary for G-protein activation. The residual activation suggests a conformer of the photolyzed pigment which mimics metarhodopsin II to a very limited extent.

Animals↗
PubMed1989

Magic angle sample spinning 13C nuclear magnetic resonance of isotopically labeled bacteriorhodopsin.

Bacteriorhodopsin (bR), the light-driven proton pump protein from Halobacterium halobium, was biosynthetically labeled with [4-13C]Asp. The incorporation yield was 48%. The magic angle sample spinning (MASS) 13C nuclear magnetic resonance (NMR) spectrum of this sample revealed six different peaks superimposed on a broad band of naturally abundant peptide-bond 13C. Two of the six carbonyl signals can be attributed to internal-protonated Asp carboxyl groups, one of which might be Asp115. An additional resonance at 110 ppm can be associated with the C-11 carbon of Trp, indicating an unusual biosynthetic pathway of this amino acid in Halobacterium halobium. Similar measurements performed on papain-treated purple membrane which lacks the C-terminal tail display two new intense signals at 178 and 178.9 ppm. If the same spectrum is taken without cross-polarization, these signals do not decrease or disappear. On the basis of their intensities and their chemical shifts, one can assign in addition to the C-terminal Asp four Asp residues facing the cytoplasmic phase. In native bR, at least two of these form a salt-bridge-like bond which also might include the C-terminal tail. These experiments not only provide data about the chemical environment of the Asp residues within the hydrophobic core of bacteriorhodopsin but also yield information about the interactions between surface components.

Amino Acid Sequence↗
PubMed1988

Rhodopsin-lumirhodopsin phototransition of bovine rhodopsin investigated by Fourier transform infrared difference spectroscopy.

The rhodopsin-lumirhodopsin transition has been investigated by Fourier transform infrared difference spectroscopy using isotope-labeled retinals. In the transition, two protonated carboxyl groups are involved. Another carbonyl band, located at 1725 cm-1 in rhodopsin, is shifted to 1731.5 cm-1 in lumirhodopsin. This line is tentatively assigned to a carbonyl stretching vibration of a peptide bond adjacent to the nitrogen of a proline residue. The C=N stretching vibration of rhodopsin could unequivocally be assigned to a band at 1659 cm-1. In contrast to rhodopsin and bathorhodopsin, the C=N stretching vibration of lumirhodopsin is at a low position, i.e., at 1635 cm-1, and exhibits only a downshift of 4 cm-1 upon deuteriation of the nitrogen. The C15-H rocking vibration of rhodopsin is assigned to the unusual high position of 1456 cm-1 and shifts into the normal region upon formation of lumirhodopsin. From these results, it is concluded that, whereas the environment of the Schiff base in rhodopsin, bathorhodopsin, and isorhodopsin is approximately the same, large changes occur with the formation of lumirhodopsin. From the assignment of the C10-C11 stretching vibration in bathorhodopsin and lumirhodopsin, a 10-s-cis geometry of lumirhodopsin can be excluded.

Animals↗
PubMed1988

The photoreaction of vacuum-dried rhodopsin at low temperature: evidence for charge stabilization by water.

The photoreaction of vacuum-dried rhodopsin was monitored by UV-visible absorption spectroscopy. The results indicate that in dry rhodopsin, isorhodopsin and lumirhodopsin a protonation equilibrium exists between the protonated and the non-protonated Schiff base. On hydration the water stabilizes the protonated forms. In metarhodopsin-I the protein itself is able to stabilize the protonated Schiff base. The direct involvement of water in the retinal binding site was demonstrated by measuring the rhodopsin-bathorhodopsin FTIR difference spectra of rhodopsin hydrated with H2O and H2(18)O. The results are discussed with respect to the problem of charge stabilization and energy storage.

Animals↗
PubMed1986

Evidence for light-induced 13-cis, 14-s-cis isomerization in bacteriorhodopsin obtained by FTIR difference spectroscopy using isotopically labelled retinals.

We have obtained by Fourier transformed infra-red (FTIR)-spectroscopy BR-K, BR-L and BR-M difference spectra of bacteriorhodopsin regenerated with isotopically labelled retinals. Thereby, we are able to assign reliably the C(14)-C(15) and C=N stretching vibrations of the various intermediates. The lower C(14)-C(15) stretching vibration frequency in L as compared with 13-cis protonated Schiff base model compounds indicates a 13-cis, 14-s-cis configuration of the retinal in this species. The unusually low C=N stretching vibration in K at 1615 cm indicates less stabilization of the positive charge at the Schiff base by the protein environment. Based on these results, a mechanism is suggested by which the stored light energy is transformed into proton transfers.

Journal Article↗
PubMed1985

Light-driven protonation changes of internal aspartic acids of bacteriorhodopsin: an investigation by static and time-resolved infrared difference spectroscopy using [4-13C]aspartic acid labeled purple membrane.

The molecular events during the photocycle of bacteriorhodopsin have been studied by the method of time-resolved and static infrared difference spectroscopy. Characteristic spectral changes involving the C=O stretching vibration of protonated carboxylic groups were detected. To identify the corresponding groups with either glutamic or aspartic acid, BR was selectively labeled with [4-13C]aspartic acid. An incorporation of ca. 70% was obtained. The comparison of the difference spectra in the region of the CO2- stretching vibrations of labeled and unlabeled BR indicates that ionized aspartic acids are influenced during the photocycle, the earliest effect being observed already at the K610 intermediate. Taken together, the results provide evidence that four internal aspartic acids undergo protonation changes and that one glutamic acid, remaining protonated, is disturbed. The results are discussed in relation to the various aspects of the proton pumping mechanism, such as retinal isomerization, charge separation, pK changes, and proton pathway.

Aspartic Acid↗
PubMed1983

Fourier-transform infrared spectroscopy applied to rhodopsin. The problem of the protonation state of the retinylidene Schiff base re-investigated.

By measuring the rhodopsin--bathorhodopsin, isorhodopsin--bathorhodopsin, rhodopsin--isorhodopsin and rhodopsin--meta-II difference spectra with the method of Fourier-transform infrared spectroscopy we have identified the C = N stretching vibration of the protonated retinylidene Schiff base of rhodopsin, isorhodopsin and bathorhodopsin. In contrast to resonance Raman spectroscopy additional strong bands were observed between 1700 cm-1 and 1620 cm-1. Most of them depend on the isomeric state of the chromophore. The origin of these bands will be discussed. In the fingerprint region isorhodopsin and bathorhodopsin are quite similar but no similarities with infrared spectra of model compounds of any isomeric composition are observed. Therefore, no conclusions on the isomeric state of the retinal in bathorhodopsin can be drawn. We provide evidence for the modification of one or two carboxylic group(s) during the rhodopsin--bathorhodopsin and isorhodopsin--bathorhodopsin transition.

Animals↗
PubMed1983

Investigation of the primary photochemistry of bacteriorhodopsin by low-temperature Fourier-transform infrared spectroscopy.

The method of Fourier-transform infrared difference spectroscopy was applied to investigate the transition at 77K of bacteriorhodopsin in its light-adapted form to K6(10), the first intermediate which is stable at low temperature. In addition to unmodified bacteriorhodopsin, bacteriorhodopsin in 2H2O and bacteriorhodopsin containing [15-2H]retinal was used. The results show that major rearrangements occur in the Schiff base in this transition. It is not possible to identify a C = N stretching vibration of the Schiff base in K6(10). The identification of an N-H bending vibration in K6(10) shows that the nitrogen of the previous Schiff base still has a proton attached. The fingerprint region exhibits very unusual features for K6(10) and bears no similarity to protonated retinylidene Schiff base model compounds of any isomeric composition. Therefore, no conclusions on the isomeric state of the retinal in K6(10) can be drawn. The spectra show that the terminal part of the retinal is predominantly reflected in the difference spectra. This indicates that the most polar part of the retinal is located near the Schiff base. We have evidence for protein molecular changes occurring in this transition at 77K.

Bacteriorhodopsins↗
PubMed1980

Flash-induced kinetic infrared spectroscopy applied to biochemical systems.

A flash photolysis apparatus with monitoring infrared beam is described allowing measurements of relative transmission changes of 10(-3) in times of a few milliseconds. The investigation of the photodissociation of CO-myoglobin confirms the results obtained by static infrared difference spectroscopy. The application of our method to the rhodopsin/Meta II transition reveals signals which can tentatively be ascribed to the disappearance of the C = C-band of the protonated N-retinylidene Schiff base in rhodopsin. The developed method will be compared with other existing methods of kinetic vibronic spectroscopy such as kinetic resonance Raman spectroscopy and kinetic Fourier infrared spectroscopy.

Animals↗
PubMed1980

Investigations of the rhodopsin/Meta I and rhodopsin/Meta II transitions of bovine rod outer segments by means of kinetic infrared spectroscopy.

We have applied our recently developed technique of flash induced kinetic infrared spectroscopy to the rhodopsin/Meta I and rhodopsin/Meta II transitions. Features of the infrared spectrum reflecting the C = C-vibration and the isomeric form of the chromophore are in agreement with resonant Raman experiments. Different results are obtained for the C = N-vibration of the Schiff base retinal opsin link. They are interpreted in terms of a Schiff base protonated via an hydrogen bond. A proton transfer in the excited state is suggested to explain the deviating results. In addition we have obtained spectral changes which cannot be attributed to molecular changes in the chromophore. We assume that these spectral features reflect molecular events in the protein part of rhodopsin.

Animals↗