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H Otto

Publications and source records attributed to H Otto.

At least 19 recordsLinked to original sources
PubMed1992

Effect of introducing different carboxylate-containing side chains at position 85 on chromophore formation and proton transport in bacteriorhodopsin.

During the initial stages of the bacteriorhodopsin photocycle, a proton is transferred from the Schiff base to the deprotonated carboxylate of Asp85. Earlier studies have shown that replacement of Asp85 by Asn completely abolishes proton transport activity, whereas extension of the side chain by an additional carbon-carbon bond (Asp85-->Glu) results in a functional proton pump. Here we show that extension of the Asp85 side chain by two additional bond lengths also results in a functional proton pump as long as the terminal group is a carboxylate moiety. These side chains were created by modification of the cysteine residue in the Asp85-->Cys mutant with either iodoacetic acid or iodoacetamide. In vitro chromophore formation studies show that the rate of Schiff base protonation in mutants that contain a carboxylate at residue 85 is invariably faster than in mutants that contain neutral substitutions at this position. We conclude that in bacteriorhodopsin, there is considerable tolerance in the volume of the side chain that can be accommodated at position 85 and that the presence of a carboxylate at residue 85 is important both for proton pumping and for stabilizing the protonated Schiff base.

Amino Acid Sequence↗
PubMed1992

Properties of protein kinase C associated with nuclear membranes.

To study signal transduction directed towards the cell nucleus and at the nuclear membranes, we investigated the association of protein kinase C (PKC) with nuclear membranes obtained from nuclei isolated from bovine brain. By use of phorbol-ester-binding assays, significant amounts of PKC could be demonstrated in nuclei and nuclear membranes. Nuclear membranes are shown to be able to activate purified PKC. The PKC endogenously present in nuclear membranes appears to be a so-called 'membrane-inserted' form: it is permanently active, still binds phorbol ester, but its activity is no longer dependent on Ca2+ and cannot be activated by phorbol ester. On the other hand, this form of PKC can be inhibited by specific PKC inhibitors. By using histone HIIIS and a specific peptide substrate, it could be shown that after extraction with Triton X-100 the PKC can be stimulated by phospholipid again. Immunoblot analysis with isoenzyme-specific antibodies revealed that the alpha- and gamma-isoenzymes, but not the beta-isoenzyme, are associated with membranes derived from brain nuclei.

Amino Acid Sequence↗
PubMed1992

Anion binding to the Schiff base of the bacteriorhodopsin mutants Asp-85----Asn/Asp-212----Asn and Arg-82----Gln/Asp-85----Asn/Asp-212----Asn.

Studies of bacteriorhodopsin have indicated that the charge environment of the protonated Schiff base consists of residues Asp-85, Asp-212, and Arg-82. As shown recently (Marti, T., Rösselet, S. J., Otto, H., Heyn, M. P., and Khorana, H. G. (1991) J. Biol. Chem. 266, 18674-18683), in the double mutant Asp-85----Asn/Asp-212----Asn chromophore formation is restored in the presence of salts, suggesting that exogenous anions function as counterions to the protonated Schiff base. To investigate the role of Arg-82 and of the Schiff base in anion binding, we have prepared the triple mutant Arg-82----Gln/Asp-85----Asn/Asp-212----Asn and compared its properties with those of the Asp-85----Asn/Asp-212----Asn double mutant. Regeneration of the chromophore with absorption maximum near 560 nm occurs in the triple mutant in the presence of millimolar salt, whereas in the double mutant molar salt concentrations are required. Spectrometric titrations reveal that the pKa of Schiff base deprotonation is markedly reduced from 11.3 for the wild type to 4.9 for the triple mutant in 1 mM NaCl and to 5.5 for the double mutant in 10 mM NaCl. In both mutants, increasing the chloride concentration promotes protonation of the chromophore and results in a continuous rise of the Schiff base pKa, yielding a value of 8.4 and 7.6, respectively, in 4 M NaCl. The absorption maximum of the two mutants shows a progressive red shift, as the ionic radius of the halide increases in the sequence fluoride, chloride, bromide, and iodide. An identical spectral correlation in the presence of halides is observed for the acid-purple form of bacteriorhodopsin. We conclude, therefore, that upon neutralization of the two counterions Asp-85 and Asp-212 by mutation or by protonation at low pH, exogenous anions substitute as counterions by directly binding to the protonated Schiff base. This interaction may provide the basis for the proposed anion translocation by the acid-purple form of bacteriorhodopsin as well as by the related halorhodopsin.

Amino Acid Sequence↗
PubMed1992

A bacteriorhodopsin analog reconstituted with a nonisomerizable 13-trans retinal derivative displays light insensitivity.

With the aim of preparing a light-insensitive bacteriorhodopsin-like pigment, bacterio-opsin expressed in Escherichia coli was treated in phospholipid-detergent micelles with the retinal analog II, in which the C13-C14 trans-double bond cannot isomerize due to inclusion in a cyclopentene ring. The formation of a complex with a fine structure (lambda max, 439 nm) was first observed. This partially converted over a period of 12 days to a bacteriorhodopsin-like chromophore (ebR-II) with lambda max, 555 nm. An identical behavior has been observed previously upon reconstitution of bleached purple membrane with the analog II. Purification by gel filtration gave pure ebR-II with lambda max, 558 nm, similar to that of light-adapted bacterio-opsin reconstituted with all-trans retinal (ebR-I). Spectrophotometric titration of ebR-II as a function of pH showed that the purple to blue transition of bacteriorhodopsin at acidic pH was altered, and the apparent pKa of Schiff base deprotonation at alkaline pH was lowered by 2.4 units, relative to that of ebR-I. ebR-II showed no light-dark adaptation, no proton pumping, and no intermediates characteristic of the bacteriorhodopsin photocycle. In addition, the rates of reaction with hydroxylamine in the dark and in the light were similar. These results show, as expected, that isomerization of the C13-C14 double bond is required for bacteriorhodopsin function and that prevention of this isomerization confers light insensitivity.

Bacteriorhodopsins↗
PubMed1992

Consequences of amino acid insertions and/or deletions in transmembrane helix C of bacteriorhodopsin.

Six bacterioopsin mutants containing either single amino acid deletions (delta A84, delta L87), insertions (delta 85A, delta 88A), or both deletions and insertions (delta A84/delta 88A, delta 85A/delta L87) within the first two turns of transmembrane helix C, starting from the extracellular side, have been prepared. The mutant apoproteins refold in phospholipid/detergent micelles and display secondary structures similar to that of the wild type. However, the mutants delta 88A and delta A84/delta 88A do not form a chromophore with retinal. The regenerated chromophore of delta 85A displays absorption maxima and retinal isomer compositions in the dark- and light-adapted states similar to those of the wild type. In delta A84, delta L87, and delta 85A/delta L87 these chromophore properties are altered, and the structures are less stable than that of the wild type, as shown by an enhanced rate of reaction with hydroxylamine in the dark, an increased pKa of the denaturation at acidic pH, and a decreased pKa of Schiff base deprotonation. Proton translocation is abolished in the delta A84 and delta 85A/delta L87 mutants, whereas in delta 85A and delta L87 the activity is reduced to about 25% of the wild-type value at pH 6. The overall properties of the delta 85A, delta 85A/delta L87, and delta L87 mutants indicate that the deletions and/or insertions result in displacement of residues Arg-82, Asp-85, or Asp-96, respectively, which participate in proton translocation. The results are compatible with a helical structure for transmembrane segment C and emphasize the flexibility of intramolecular contacts in bacteriorhodopsin.

Amino Acid Sequence↗
PubMed1992

GTP-binding proteins in bovine brain nuclear membranes.

Nuclear membranes and other subcellular fractions derived from bovine brain cortex were investigated for the existence of GTP-binding proteins. By using photolytic labeling with [alpha-32P]GTP a 29 kDa GTP-binding protein was shown to be present in nuclear membranes which was not present in the plasma membranes nor in microsomal or cytosolic fractions. Two-dimensional gel electrophoresis revealed that this protein is rather acidic with a pI lower than 4.5. Members of the heterotrimeric Gi/o family are not present in the nuclear envelope: a 39 kDa protein, ADP ribosylated by pertussis toxin, was shown to originate from plasma membrane contamination.

Adenosine Diphosphate Ribose↗
PubMed1991

Between the ground- and M-state of bacteriorhodopsin the retinal transition dipole moment tilts out of the plane of the membrane by only 3 degrees.

The orientation of the transition dipole moments in the ground state and the M-intermediate of bacteriorhodopsin were determined by time-resolved and steady-state polarized absorption spectroscopy on samples of oriented immobilized purple membranes. The angle between the transition dipole moment and the membrane normal decreases from 66.8 +/- 0.5 degrees in the all-trans ground state to 64.1 +/- 0.8 degrees in the 13-cis M-state. The light-induced isomerization of the chromophore is thus accompanied by an orientational change of only about 3 degrees out of the plane of the membrane. The absorption anisotropy at 410 nm remains constant over more than 4 decades of time covering both the rise and decay of M. Conformational changes accompanying a sequential M1----M2 transition thus do not affect the chromophore orientation.

Bacteriorhodopsins↗
PubMed1991

The retinylidene Schiff base counterion in bacteriorhodopsin.

Previous studies of bacteriorhodopsin have indicated interactions between Asp-85, Asp-212, Arg-82, and the retinylidene Schiff base. The counterion environment of the Schiff base has now been further investigated by using single and double mutants of the above amino acids. Chromophore regeneration from bacterioopsin proceeds to a normal extent in the presence of a single aspartate or glutamate residue at position 85 or 212, whereas replacement of both charged amino acids in the mutant Asp-85----Asn/Asp-212----Asn abolishes the binding of retinal. This indicates that a carboxylate group at either residue 85 or 212 is required as counterion for formation and for stabilization of the protonated Schiff base. Measurements of the pKa of the Schiff base reveal reductions of greater than 3.5 units for neutral single mutants of Asp-85 but only decreases of less than 1.2 units for corresponding substitutions of Asp-212, relative to the wild type. Substitutions of Asp-85 show large red shifts in the absorption spectrum that are partially reversible upon addition of anions, whereas mutants of Asp-212 display minor red shifts or blue shifts. We conclude, therefore, that Asp-85 is the retinylidene Schiff base counterion in wild-type bacteriorhodopsin. In the mutant Asp-85----Asn/Asp-212----Asn formation of a protonated Schiff base chromophore is restored in the presence of salts. The spectral properties of the double mutant are similar to those of the acid-purple form of bacteriorhodopsin. Upon addition of salts the folded structure of wild-type and mutant proteins can be stabilized at low pH in lipid/detergent micelles. The data indicate that exogenous anions serve as surrogate counterions to the protonated Schiff base, when the intrinsic counterions have been neutralized by mutation or by protonation.

Bacteriorhodopsins↗
PubMed1991

Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation.

To study their role in proton translocation by bacteriorhodopsin, 22 serine and threonine residues presumed to be located within and near the border of the transmembrane segments have been individually replaced by alanine or valine, respectively. Thr-89 was substituted by alanine, valine, and aspartic acid, and Ser-141 by alanine and cysteine. Most of the mutants showed essentially wild-type phenotype with regard to chromophore regeneration and absorption spectrum. However, replacement of Thr-89 by Val and of Ser-141 by Cys caused striking blue shifts of the chromophore by 100 and 80 nm, respectively. All substitutions of Thr-89 regenerated the chromophore at least 10-fold faster with 13-cis retinal than with all-trans retinal. The substitutions at positions 89, 90, and 141 also showed abnormal dark-light adaptation, suggesting interactions between these residues and the retinylidene chromophore. Proton pumping measurements revealed 60-75% activity for mutants of Thr-46, -89, -90, -205, and Ser-226, and about 20% for Ser-141----Cys, whereas the remaining mutants showed normal pumping. Kinetic studies of the photocycle and of proton release and uptake for mutants in which proton pumping was reduced revealed generally little alterations. The reduced activity in several of these mutants is most likely due to a lower percentage of all-trans retinal in the light-adapted state. In the mutants Thr-46----Val and Ser-226----Ala the decay of the photointer-mediate M was significantly accelerated, indicating an interaction between these residues and Asp-96 which reprotonates the Schiff base. Our results show that no single serine or threonine residue is obligatory for proton pumping.

Amino Acid Sequence↗
PubMed1990

Transmembrane location of retinal in bacteriorhodopsin by neutron diffraction.

The transmembrane location of the chromophore of bacteriorhodopsin was obtained by neutron diffraction on oriented stacks of purple membranes. Two selectively deuterated retinals were synthesized and incorporated in bacteriorhodopsin by using the retinal- mutant JW5: retinal-d11 (D11) contained 11 deuterons in the cyclohexene ring, and retinal-d5 (D5) had 5 deuterons as close as possible to the Schiff base end of the chromophore. The membrane stacks had a lamellar spacing of 53.1 A at 86% relative humidity. Five orders were observed in the lamellar diffraction pattern of the D11, D5, and nondeuterated reference samples. The reflections were phased by D2O-H2O exchange. The absolute values of the structure factors were nonlinear functions of the D2O content, suggesting that the coherently scattering domains consisted of asymmetric membrane stacks. The centers of deuteration were determined from the observed intensity differences between labeled and unlabeled samples by using model calculations and Fourier difference methods. With the origin of the coordinate system defined midway between consecutive intermembrane water layers, the coordinates of the center of deuteration of the D11 and D5 label are 10.5 +/- 1.2 and 3.8 +/- 1.5 A, respectively. Alternatively, the label distance may be measured from the nearest membrane surface as defined by the maximum in the neutron scattering length density at the water/membrane interface. With respect to this point, the D11 and D5 labels are located at a depth of 9.9 +/- 1.2 and 16.6 +/- 1.5 A, respectively. The chromophore is tilted with the Schiff base near the middle of the membrane and the ring closer to the membrane surface. The vector connecting the two label positions in the chromophore makes an angle of 40 +/- 12 degrees with the plane of the membrane. Of the two possible orientations of the plane of the chromophore, which is perpendicular to the membrane plane, only the one in which the N----H bond of the Schiff base points toward the same membrane surface as the vector from the Schiff base to the cyclohexene ring is compatible with the known tilt angle of the polyene chain.

Bacteriorhodopsins↗
PubMed1990

Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base.

Photocycle and flash-induced proton release and uptake were investigated for bacteriorhodopsin mutants in which Asp-85 was replaced by Ala, Asn, or Glu; Asp-212 was replaced by Asn or Glu; Asp-115 was replaced by Ala, Asn, or Glu; Asp-96 was replaced by Ala, Asn, or Glu; and Arg-82 was replaced by Ala or Gln in dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate micelles at pH 7.3. In the Asp-85----Ala and Asp-85----Asn mutants, the absence of the charged carboxyl group leads to a blue chromophore at 600 and 595 nm, respectively, and lowers the pK of the Schiff base deprotonation to 8.2 and 7, respectively, suggesting a role for Asp-85 as counterion to the Schiff base. The early part of the photocycles of the Asp-85----Ala and Asp-85----Asn mutants is strongly perturbed; the formation of a weak M-like intermediate is slowed down about 100-fold over wild type. In both mutants, proton release is also slower but clearly precedes the rise of M. The amplitude of the early (less than 0.2 microseconds) reversed photovoltage component in the Asp-85----Asn mutant is very large, and the net charge displacement is close to zero, indicating proton release and uptake on the cytoplasmic side of the membrane. The data suggest an obligatory role for Asp-85 in the efficient deprotonation of the Schiff base and in the proton release phase, probably as proton acceptor. In the Asp-212----Asn mutant, the rise of the absorbance change at 410 nm is slowed down to 220 microsecond, its amplitude is small, and the release of protons is delayed to 1.9 ms. The absorbance changes at 650 nm indicate perturbations in the early time range with a slow K intermediate. Thus Asp-212 also participates in the early events of charge translocation and deprotonation of the Schiff base. In the Arg-82----Gln mutant, no net transient proton release was observed, whereas, in the Arg-82----Ala mutant, uptake and release were reversed. The pK shift of the purple-to-blue transition in the Asp-85----Glu, Arg-82----Ala, and Arg-82----Gln mutants and the similarity in the photocycle and photoelectrical signals of the Asp-85----Ala, Asp-85----Asn, and Asp-212----Asn mutants suggest the interaction between Asp-85, Arg-82, Asp-212, and the Schiff base as essential for proton release.

Arginine↗
PubMed1989

The purple to blue transition of bacteriorhodopsin is accompanied by a loss of the hexagonal lattice and a conformational change.

X-ray diffraction measurements show that in contrast to the purple membrane, the bacteriorhodopsin molecules are not organized in a hexagonal lattice in the deionized blue membrane. Addition of Ca2+ restores both the purple color and the normal (63 A) hexagonal protein lattice. In the blue state, the circular dichroism spectrum in the visible has the typical exciton features indicating that a trimeric structure is retained. Time-resolved linear dichroism measurements show that the blue patch rotates in aqueous suspension with a mean correlation time of 11 ms and provide no evidence for rotational mobility of bacteriorhodopsin within the membrane. The circular dichroism spectra of the blue and the Ca2+-regenerated purple state in the far-UV are different, indicating a small change in secondary structure. The thermal stability of the blue membrane is much smaller than that of the purple membrane. At pH 5.0, the irreversible denaturation transition of the blue form has a midpoint at 61 degrees C. The photocycle of the blue membrane (lambda ex 590 nm) has an L intermediate around 540 nm whose decay is slowed down into the millisecond time range (5 ms). Light-dark adaptation in the blue membrane is rapid with an exponential decay time of 38 s at 25 degrees C. The purple to blue transition apparently involves a conformational change in the protein leading to a change in the aggregation state from a highly ordered and stable hexagonal lattice to a disordered array of thermally more labile trimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsins↗