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J Heberle

Publications and source records attributed to J Heberle.

At least 19 recordsLinked to original sources
PubMed2004

Functional variations among LOV domains as revealed by FT-IR difference spectroscopy.

The two LOV domains, LOV1 and LOV2, from Chlamydomonas reinhardtii were investigated by light-induced FT-IR difference spectroscopy and compared to the LOV domain of Bacillus subtilis (YtvA-LOV). It is shown that the two S-H conformations of the reactive LOV1 cysteine C57(1) are exposed to environments of different hydrogen bonding strength. Thus, the two rotamer configurations of C57 might be related to the fact that the triplet state decays bi-exponentially into the LOV1-390 photoproduct. Exchange of the two other cysteines of LOV1 (C32S and C83S) does not alter the S-H stretching band providing evidence that this band feature arises solely from C57. The reactive cysteine of LOV2 from Chlamydomonas reinhardtii (C250) and of YtvA-LOV (C62) exhibit both a homogenous S-H stretching vibrational band which suggests a single conformer of the amino acid side chain. Finally, the FT-IR difference spectrum of YtvA from Bacillus subtilis comprising the light absorbing LOV domain and the putative signaling STAS (sulfate transporter/antisigma-factor antagonist) domain, reveals conformational changes in the latter after blue-light excitation.

Animals↗
PubMed2003

Vibrational spectroscopy of an algal Phot-LOV1 domain probes the molecular changes associated with blue-light reception.

The LOV1 domain of the blue light Phot1-receptor (phototropin homolog) from Chlamydomonas reinhardtii has been studied by vibrational spectroscopy. The FMN modes of the dark state of LOV1 were identified by preresonance Raman spectroscopy and assigned to molecular vibrations. By comparing the blue-light-induced FTIR difference spectrum with the preresonance Raman spectrum, most of the differences are due to FMN modes. Thus, we exclude large backbone changes of the protein that might occur during the phototransformation of the dark state LOV1-447 into the putative signaling state LOV1-390. Still, the presence of smaller amide difference bands cannot be excluded but may be masked by overlapping FMN modes. The band at 2567 cm(-1) is assigned to the S-H stretching vibration of C57, the residue that forms the transient thio-adduct with the chromophore FMN. The occurrence of this band is evidence that C57 is protonated in the dark state of LOV1. This result challenges conclusions from the homologous LOV2 domain from oat that the thiolate of the corresponding cysteine is the reactive species.

Animals↗
PubMed2002

Proton translocation by bacteriorhodopsin in the absence of substantial conformational changes.

Unlike wild-type bacteriorhodopsin (BR), the BR triple mutant D96G/F171C/F219L has been shown to undergo only minor structural rearrangements during its photocycle. Nonetheless, the mutant is capable of transporting protons at a rate of 125(+/-40) H+/BR per minute under light-saturating conditions. Light adaptation of the triple mutant's retinal proceeds in a pH-dependent manner up to a maximum of 63% all-trans. These two findings imply that the transport activity of the triple mutant comprises 66% of the wild-type activity. Time-resolved spectroscopy reveals that the identity and sequence of intermediates in the photocycle of the triple mutant in the all-trans configuration correspond to that of wild-type BR. The only differences relate to a slower rise and decay of the M and O intermediates, and a significant spectral contribution from a 13-cis component. No indication for accumulation of the N intermediate is found under a variety of conditions that normally favor the formation of this species in wild-type BR. The Fourier transform infrared (FTIR) spectrum of the M intermediate in the triple mutant resembles that of wild type. Minor changes in the amide I region during the photocycle suggest that only small movements of the protein backbone occur. Electron microscopy reveals large differences in conformation between the unilluminated state of the mutant protein and wild-type but no light-induced changes in time-resolved measurements. Evidently, proton transport by the triple mutant does not require the major conformational rearrangements that occur on the same time-scale with wild-type. Thus, we conclude that large conformational changes observed in the photocycle of the wild-type and many BR mutants are not a prerequisite for the change in accessibility of the Schiff base nitrogen atom that must occur during vectorial catalysis to allow proton transport.

Archaea↗
PubMed2001

Perfusion-induced redox differences in cytochrome c oxidase: ATR/FT-IR spectroscopy.

Attenuated total reflection (ATR) spectroscopy brings an added dimension to studies of structural changes of cytochrome c oxidase (CcO) because it enables the recording of reaction-induced infrared difference spectra under a wide variety of controlled conditions (e.g. pH and chemical composition), without relying on light or potentiometric changes to trigger the reaction. We have used the ATR method to record vibrational difference spectra of CcO with reduction induced by flow-exchange of the aqueous buffer. Films of CcO prepared from Rhodobacter sphaeroides and beef heart mitochondria by reconstitution with lipid were adhered to the internal reflection element of the ATR device and retained their full functionality as evidenced by visible spectroscopy and time-resolved vibrational spectroscopy. These results demonstrate that the technique of perfusion-induced Fourier-transform infrared difference spectroscopy can be successfully applied to a large, complex enzyme, such as CcO, with sufficient signal/noise to probe vibrational changes in individual residues of the enzyme under various conditions.

Animals↗
PubMed2001

Time-resolved FT-IR spectroscopic investigation of the pH-dependent proton transfer reactions in the E194Q mutant of bacteriorhodopsin.

The photoreaction of the E194Q mutant of bacteriorhodopsin has been investigated at various pH values by time-resolved step-scan Fourier-transform infrared difference spectroscopy employing the attenuated total reflection technique. The difference spectrum at pH 8.4 is comparable to the N-BR difference spectra of the wild type with the remarkable exception that D85 is deprotonated. Since the retinal configuration is not perturbed by the E194Q mutation, it is concluded that there is no interaction of D85 with retinal during the lifetime of the N state. At pH 6, a consecutive state to the O intermediate is detected in which D212 is transiently protonated. The comparison with wild-type bacteriorhodopsin reveals that protonation of D212 represents an intermediate step during proton transfer from D85 to the proton release group in the final stage of the reaction cycle. The described effects are more pronounced in the E194Q mutant than in the E204Q mutant demonstrating different roles of these two glutamates/glutamic acids at least in the final stages of the catalytic cycle of bacteriorhodopsin.

Acids↗
PubMed2000

Bacteriorhodopsin: the functional details of a molecular machine are being resolved

The photon-driven proton translocator bacteriorhodopsin is considered to be the best understood membrane protein so far. It is nowadays regarded as a model system for photosynthesis, ion pumps and seven transmembrane receptors. The profound knowledge came from the applicability of a variety of modern biophysical techniques which have often been further developed with research on bacteriorhodopsin and have delivered major contributions also to other areas. Most prominent examples are electron crystallography, solid-state NMR spectroscopy and time-resolved vibrational spectroscopy. The recently introduced method of crystallising a membrane protein in the lipidic cubic phase led to high-resolution structures of ground state bacteriorhodopsin and some of the photocycle intermediates. This achievement in combination with spectroscopic results will strongly advance our understanding of the functional mechanism of bacteriorhodopsin on the atomic level. We present here the current knowledge on specific aspects of the structural and functional dynamics of the photoreaction of bacteriorhodopsin with a focus on techniques established in our institute.

Journal Article↗
PubMed2000

Proton transfer reactions across bacteriorhodopsin and along the membrane.

Bacteriorhodopsin is probably the best understood proton pump so far and is considered to be a model system for proton translocating membrane proteins. The basis of a molecular description of proton translocation is set by having the luxury of six highly resolved structural models at hand. Details of the mechanism and reaction dynamics were elucidated by a whole variety of biophysical techniques. The current molecular picture of catalysis by BR will be presented with examples from time-resolved spectroscopy. FT-IR spectroscopy monitors single proton transfer events within bacteriorhodopsin and judiciously positioned pH indicators detect proton migration at the membrane surface. Emerging properties are briefly outlined that underlie the efficient proton transfer across and along biological membranes.

Bacteriorhodopsins↗
PubMed2000

Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure.

We have searched for a minimal interaction motif in tau protein that supports the aggregation into Alzheimer-like paired helical filaments. Digestion of the repeat domain with different proteases yields a GluC-induced fragment comprising 43 residues (termed PHF43), which represents the third repeat of tau plus some flanking residues. This fragment self assembles readily into thin filaments without a paired helical appearance, but these filaments are highly competent to nucleate bona fide PHFs from full-length tau. Probing the interactions of PHF43 with overlapping peptides derived from the full tau sequence yields a minimal hexapeptide interaction motif of (306)VQIVYK(311) at the beginning of the third internal repeat. This motif coincides with the highest predicted beta-structure potential in tau. CD and Fourier transform infrared spectroscopy shows that PHF43 acquires pronounced beta structure in conditions of self assembly. Point mutations in the hexapeptide region by proline-scanning mutagenesis prevent the aggregation. The data indicate that PHF assembly is initiated by a short fragment containing the minimal interaction motif forming a local beta structure embedded in a largely random-coil protein.

Alzheimer Disease↗
PubMed2000

Structural equilibrium fluctuations in mesophilic and thermophilic alpha-amylase.

By comparing a mesophilic alpha-amylase with its thermophilic homolog, we investigated the relationship between thermal stability and internal equilibrium fluctuations. Fourier transform infrared spectroscopy monitoring hydrogen/deuterium (H/D) exchange kinetics and incoherent neutron scattering measuring picosecond dynamics were used to study dynamic features of the folded state at room temperature. Fairly similar rates of slowly exchanging amide protons indicate about the same free energy of stabilization DeltaG(stab) for both enzymes at room temperature. With respect to motions on shorter time scales, the thermophilic enzyme is characterized by an unexpected higher structural flexibility as compared to the mesophilic counterpart. In particular, the picosecond dynamics revealed a higher degree of conformational freedom for the thermophilic alpha-amylase. The mechanism proposed for increasing thermal stability in the present case is characterized by entropic stabilization and by flattening of the curvature of DeltaG(stab) as a function of temperature.

Bacillus↗
PubMed1999

In situ determination of transient pKa changes of internal amino acids of bacteriorhodopsin by using time-resolved attenuated total reflection Fourier-transform infrared spectroscopy.

Active proton transfer through membrane proteins is accomplished by shifts in the acidity of internal amino acids, prosthetic groups, and water molecules. The recently introduced step-scan attenuated total reflection Fourier-transform infrared (ATR/FT-IR) spectroscopy was employed to determine transient pKa changes of single amino acid side chains of the proton pump bacteriorhodopsin. The high pKa of D96 (>12 in the ground state) drops to 7.1 +/- 0.2 (in 1 M KCl) during the lifetime of the N intermediate, quantitating the role of D96 as the internal proton donor of the retinal Schiff base. We conclude from experiments on the pH dependence of the proton release reaction and on point mutants where each of the glutamates on the extracellular surface has been exchanged that besides D85 no other carboxylic group changes its protonation state during proton release. However, E194 and E204 interact with D85, the primary proton acceptor of the Schiff base proton. The C==O stretching vibration of D85 undergoes a characteristic pH-dependent shift in frequency during the M state of wild-type bacteriorhodopsin with a pKa of 5.2 (+/-0.3) which is abolished in the single-site mutants E194Q and E204Q and the quadruple mutant E9Q/E74Q/E194Q/E204Q. The double mutation E9Q/E74Q does not affect the lifetime of the intermediates, ruling out any participation of these residues in the proton transfer chain of bacteriorhodopsin. This study demonstrates that transient changes in acidity of single amino acid residues can be quantified in situ with infrared spectroscopy.

Amino Acids↗
PubMed1998

Assessing the functionality of a membrane protein in a three-dimensional crystal.

Hexagonal microcrystals of bacteriorhodopsin embedded in a lipidic cubic phase have been investigated by time-resolved FT-IR and resonance Raman spectroscopy. Retinal isomerization, conformational changes in the protein backbone and proton translocation are virtually indistinguishable from those in the native membrane. The protein is thus fully active in three-dimensional crystals.

Amides↗
PubMed1996

D38 is an essential part of the proton translocation pathway in bacteriorhodopsin.

At present, almost no knowledge exists about the functional relevance of the amino acid residues at the cytoplasmic (CP) surface of the light-driven proton pump bacteriorhodopsin (BR) although a prerequisite for efficient vectorial proton translocation is the efficient capture of protons from the alkaline cytoplasm of the cell. To identify residues involved in the proton transfer reaction steps in the CP part of BR, the aspartic and glutamic amino acids D36, D38, D102, D104, and E161 were replaced by cysteine and arginine (i.e., a negatively charged residue by a neutral or positive one at the pH of investigation). The effect of these replacements on the photo- and transport cycle was examined by time-resolved visible and infrared spectroscopy, biochemical modification studies, and activity assays in intact cells. Of the five CP amino acids studied, only the replacement of D38 resulted in severe alterations of the reaction steps in BR during the second half of the photocycle. Our data show that D38, which seemed to be a freely accessible CP surface residue lacking functional importance, is an essential part of the CP proton uptake pathway connecting the membrane surface with the Schiff base of BR, probably as the first amino acid residue at the CP entrance. D38 influences the late steps in the functional cycle, such as the occurrence of the intermediates N and O, the modulation of the hydrogen-network, the conformational changes in the protein moiety, and the deprotonation/reprotonation of D96. Opposed to this function, the surface-exposed amino acids D36, D102, D104, and E161 seem to efficiently collect protons from the aqueous bulk phase and funnel them to the entrance of the CP proton pathway.

Amino Acid Sequence↗
PubMed1994

Photoactive mitochondria: in vivo transfer of a light-driven proton pump into the inner mitochondrial membrane of Schizosaccharomyces pombe.

The light-driven proton pump bacteriorhodopsin (bR) from Halobacterium salinarium has been genetically transferred into the inner mitochondrial membrane (IM) of the eukaryotic cell Schizosaccharomyces pombe, where the archaebacterial proton pump replaces or increases the proton gradient usually formed by the respiratory chain. For targeting and integration, as well as for the correct orientation of bR in the IM, the bacterioopsin gene (bop) was fused to signal sequences of IM proteins. Northern and Western blot analysis proved that all hybrid gene constructs containing the bop gene and a mitochondrial signal sequence were expressed and processed to mature bR. Fast transient absorption spectroscopy showed photocycle activity of bR integrated in the IM by formation of the M intermediate. Experiments with the pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein revealed bR-mediated proton pumping from the mitochondrial matrix into the intermembrane space. Glucose uptake measurements under anaerobic conditions showed that yeast cells containing photoactive mitochondria need less sugar under illumination. In summary, our experiments demonstrate the functional genetic transfer of a light energy converter to a naturally nonphotoactive eukaryotic organism.

Bacteriorhodopsins↗
PubMed1994

Proton migration along the membrane surface and retarded surface to bulk transfer.

Since the proposal of the chemiosmotic theory there has been a continuing debate about how protons that have been pumped across membranes reach another membrane protein that utilizes the established pH gradient. Evidence has been gathered in favour of a 'delocalized' theory, in which the pumped protons equilibrate with the aqueous bulk phase before being consumed, and a 'localized' one, in which protons move exclusively along the membrane surface. We report here that after proton release by an integral membrane protein, long-range proton transfer along the membrane surface is faster than proton exchange with the bulk water phase. The rate of lateral proton diffusion can be calculated by considering the buffer capacity of the membrane surface. Our results suggest that protons can efficiently diffuse along the membrane surface between a source and a sink (for example H(+)-ATP synthase) without dissipation losses into the aqueous bulk.

Arylsulfonates↗
PubMed1993

Bacteriorhodopsin expressed in Schizosaccharomyces pombe pumps protons through the plasma membrane.

Bacterioopsin (bO) from Halobacterium salinarium ("Halobacterium halobium") has been functionally expressed in a heterologous system, the fission yeast Schizosaccharomyces pombe. Regeneration of bO to bacteriorhodopsin (bR) in S. pombe has been achieved in vivo by addition of the chromophore retinal to the culture medium, as shown for a retinal-negative mutant of H. salinarium (JW5). Western blot analysis revealed that bR is more stable than bO against proteolysis in fission yeast and also in JW5. The light-driven proton pump is expressed in the eukaryotic organism and incorporated into the plasma membrane. Illumination of intact yeast cells leads to acidification of the external medium due to the translocation of H+ from inside to outside of the cell, indicating the same orientation of bR in the yeast plasma membrane as in H. salinarium. The kinetics of proton release into the water phase was observed with the optical pH indicator pyranine. Time-resolved absorbance changes of isolated plasma membrane measured by flash spectroscopy showed rise and decay of the M intermediate during the photocycle similar to those in the homologous system. Photocurrents and photovoltages were recorded with yeast plasma membrane attached to a planar lipid membrane and to a polytetrafluoroethylene (Teflon) film, respectively. Stationary currents measured in the presence of a protonophore showed continuous pumping activity of bR. The action spectrum of the photocurrent and the kinetics of the photovoltage were analyzed and compared with signals obtained from purple membranes. From all these different investigations we conclude that the integral membrane protein bR is correctly folded in vivo into the cytoplasmic membrane of the fission yeast S. pombe.

Bacteriorhodopsins↗
PubMed1993

Decoupling of photo- and proton cycle in the Asp85-->Glu mutant of bacteriorhodopsin.

Surface bound pH indicators were applied to study the proton transfer reactions in the mutant Asp85-->Glu of bacteriorhodopsin in the native membrane. The amino acid replacement induces a drastic acceleration of the overall rise of the M intermediate. Instead of following this acceleration, proton ejection to the extracellular membrane surface is not only two orders of magnitude slower than M formation, it is also delayed as compared with the wild-type. This demonstrates that Asp85 not only accepts the proton released by the Schiff's base but also regulates very efficiently proton transfer within the proton release chain. Furthermore, Asp85 might be the primary but is not the only proton acceptor/donor group in the release pathway. The Asp85-->Glu substitution also affects the proton reuptake reaction at the cytoplasmic side, although Asp85 is located in the proton release pathway. Proton uptake is slower in the mutant than in the wild-type and occurs during the lifetime of the O intermediate. This demonstrates a feed-back mechanism between Asp85 and the proton uptake pathway in bacteriorhodopsin.

Asparagine↗