Dieter Oesterhelt

Electron diffraction analysis of structural changes in the photocycle of bacteriorhodopsin.

Structural changes are central to the mechanism of light‐driven proton transport by bacteriorhodopsin, a seven‐helix membrane protein. The main intermediate formed upon light absorption is M, which occurs between the proton release and uptake steps of the photocycle. To investigate the structure of the M intermediate, we have carried out electron diffraction studies with two‐dimensional crystals of wild‐type bacteriorhodopsin and the Asp96‐‐>Gly mutant. The M intermediate was trapped by rapidly freezing the crystals in liquid ethane following illumination with a xenon flash lamp at 5 and 25 degrees C. Here, we present 3.5 A resolution Fourier projection maps of the differences between the M intermediate and the ground state of bacteriorhodopsin. The most prominent structural changes are observed in the vicinity of helices F and G and are localized to the cytoplasmic half of the membrane.

Bacteriorhodopsin: a biological material for information processing.

Technology which makes use of biological materials has advanced dramatically in the last few decades. Production of specific biochemicals by selected microbial strains, the use of enzymes for stereospecific biosynthesis of materials and gene technological production of biologically important macromolecules are a few examples of these developments.

The structure and mechanism of the family of retinal proteins from halophilic archaea.

Retinal proteins from halophilic archaea provide a unique opportunity to analyze vectorial ion translocation. Studies on its structure, conformational changes, proton conduction and electrogenic steps have helped to elucidate the catalytic cycle of bacteriorhodopsin in increasing detail. Experimental modulation of the vectoriality and ion specificity by altering the substrate availability, point mutations and light conditions for the different retinal proteins allows the proposal of a general model of ion transport for this protein family.

The genome of the square archaeon Haloquadratum walsbyi : life at the limits of water activity

BackgroundThe square halophilic archaeon Haloquadratum walsbyi dominates NaCl-saturated and MgCl2 enriched aquatic ecosystems, which imposes a serious desiccation stress, caused by the extremely low water activity. The genome sequence was analyzed and physiological and physical experiments were carried out in order to reveal how H. walsbyi has specialized into its narrow and hostile ecological niche and found ways to cope with the desiccation stress.ResultsA rich repertoire of proteins involved in phosphate metabolism, phototrophic growth and extracellular protective polymers, including the largest archaeal protein (9159 amino acids), a homolog to eukaryotic mucins, are amongst the most outstanding features. A relatively low GC content (47.9%), 15–20% less than in other halophilic archaea, and one of the lowest coding densities (76.5%) known for prokaryotes might be an indication for the specialization in its unique environmentConclusionAlthough no direct genetic indication was found that can explain how this peculiar organism retains its square shape, the genome revealed several unique adaptive traits that allow this organism to thrive in its specific and extreme niche.

Observation of a bacteriochlorophyll anion radical during the primary charge separation in a reaction center

The primary light-induced charge separation in reaction centers of Rhodobacter sphaeroides was investigated with femtosecond time resolution. The absorption changes in the time range 100 fs to 1 ns observed after direct excitation of the primary donor P at 860 nm could only be explained by a kinetic model which uses three time constants. This finding supports the following reaction scheme: (i) the electronically excited primary donor P* decays with a time constant of 3.5 ps and populates a very short-lived intermediate involving a reduced accessory bacteriochlorophyll molecule; (ii) with a time constant of 0.9 ps the electron is transferred to the neighboring bacteriopheophytin molecule; and (iii) from there within 200 ps to the quinone.

The 'light' and 'medium' subunits of the photosynthetic reaction centre from Rhodopseudomonas viridis: isolation of the genes, nucleotide and amino acid sequence.

The ‘light’ (L) and the ‘medium’ (M) subunits of the photosynthetic reaction centre from Rhodopseudomonas viridis were isolated and their amino‐terminal sequences, as well as the sequences of several chymotryptic peptides, determined. Rps. viridis DNA was cloned in the Escherichia coli plasmid pBR322. Mixed oligonucleotide probes derived from the amino acid sequences were synthesised and utilised to isolate one clone which contained the genes for the L and M subunits of the reaction centre as well as the α and β subunits of the light‐harvesting complex and part of the gene for the reaction centre cytochrome. The nucleotide sequences of the L and M subunit genes and the derived amino acid sequences are presented. The L subunit consists of 273 amino acids and has a mol. wt of 30 571. The M subunit consists of 323 amino acids and has a mol. wt of 35 902. The primary structure is discussed in the light of the recently published secondary and tertiary structure which has shown that both subunits contain five membrane‐spanning helices.

Time-resolved X-ray diffraction study of structural changes associated with the photocycle of bacteriorhodopsin.

The time course of structural changes accompanying the transition from the M412 intermediate to the BR568 ground state in the photocycle of bacteriorhodopsin (BR) from Halobacterium halobium was studied at room temperature with a time resolution of 15 ms using synchrotron radiation X‐ray diffraction. The M412 decay rate was slowed down by employing mutated BR Asp96Asn in purple membranes at two different pH‐values. The observed light‐induced intensity changes of in‐plane X‐ray reflections were fully reversible. For the mutated BR at neutral pH the kinetics of the structural alterations (tau 1/2 = 125 ms) were very similar to those of the optical changes characterizing the M412 decay, whereas at pH 9.6 the structural relaxation (tau 1/2 = 3 s) slightly lagged behind the absorbance changes at 410 nm. The overall X‐ray intensity change between the M412 intermediate and the ground state was about 9% for the different samples investigated and is associated with electron density changes close to helix G, B and E. Similar changes (tau 1/2 = 1.3–3.6 s), which also confirm earlier neutron scattering results on the BR568 and M412 intermediates trapped at ‐180 degrees C, were observed with wild type BR retarded by 2 M guanidine hydrochloride (pH 9.4). The results unequivocally prove that the tertiary structure of BR changes during the photocycle.

Excited-state reaction dynamics of bacteriorhodopsin studied by femtosecond spectroscopy

The photodynamics of bacteriorhodopsin were studied by transient absorption and gain measurements after excitation with femtosecond pulses at 620 nm. With probing pulses at longer wavelengths (λ > 770 nm) the previously reported formation of the J intermediate (with a time constant of 500±100 fs) was confirmed. With probing pulses around 700 nm, a faster process with a relaxation time of 200±70 fs was observed. The data analysis strongly suggests that this kinetic constant describes the reactive motion of the polyatomic molecule on its excited-state potential energy surface, i.e. one observes directly the incipient isomerization of the retinal molecule. The minimum of the S1 potential energy surface reached in 200 fs lies approximately 13300 cm−1 above the ground state of bacteriorhodopsin and from this minimum the intermediate J is formed with a time constant of 500 fs.

Aspartic acids 96 and 85 play a central role in the function of bacteriorhodopsin as a proton pump.

A spectroscopic and functional analysis of two point‐mutated bacteriorhodopsins (BRs) from phototrophic negative halobacterial strains is reported. Bacteriorhodopsin from strain 384 contains a glutamic acid instead of an aspartic acid at position 85 and BR from strain 326 contains asparagine instead of aspartic acid at position 96. Compared to wild‐type BR, the M formation in BR Asp85–‐Glu is accwelerated approximately 10‐fold, whereas the M decay in BR Asp96–‐Asn is slowed down approximately 50‐fold at pH6. Purple membrane sheets containing the mutated BRs were oriented and immobilized in polyacrylamide gels or adsorbed to planar lipid films. The measured kinetics of the photocurrents under various conditions agree with the observed photocycle kinetics. The ineffectivity of BR Asp85–‐Glu resides in the dominance of an inactive species absorbing maximally at approximately 610 nm, while BR Asp96–‐Asn is ineffective due to its slow photocycle. These experimental results suggest that aspartic acid 96 plays a crucial role for the reprotonation of the Schiff base. Both residues are essential for an effective proton pump.

Chromophore equilibria in bacteriorhodopsin.

An investigation of the dark equilibria between different chromophores of bacteriorhodopsin (BR) and studies of the kinetics of their interconversion and photochemical activity have led to the following conclusions. (a) A component of the 605-nm chromophore of BR decays in the millisecond range and is likely to be identical to the intermediate O of the photochemical cycle of BR and is assumed to be formed from the purple complex (PC) by the binding of one proton to BR. (b) An acidic form the PC, PCaL-, arises from the 605-nm chromophore by selective binding of anions L- (F- greater than Cl- greater than Br- greater than I- greater than Cl04-) to BR. (c) The isomeric equilibrium between 13-cis and all-trans retinal is approximately 0.15/0.85 in PCaCl-, 0.3/0.7 in the 605-nm chromophore as compared to 0.5/0.5 in the PC. (d) The 500-nm chromophore is formed from the PC by release of nearly one proton from BR. (e) The pH range in which the PC exists is reduced in a high-temperature structure of the purple membrane as compared to its low temperature structure. A model for the chromophore structure is proposed as a hypothesis, which allows a comprehensive interpretation of the results. In this model the absorption spectrum of the retinylidene lysine Schiff base is modulated by its protonation state and the interaction with an anionic group.