Insight into a single halobacterium using a dual‐bacteriorhodopsin system with different functionally optimized pH ranges to cope with periplasmic pH changes associated with continuous light illumination

The light‐driven outward proton transporter assists energy production via an ATP synthase system best exemplified by the bacteriorhodopsin (BR) from Halobacterium salinarum, HsBR. As the only archaea able to survive in the resource‐limited ecosystem of the Dead Sea, Haloarcula marismortui has been reported to have a unique dual‐BR system, consisting of HmBRI and HmBRII, instead of only a single BR in a cell (solo‐BR). The contribution of this dual‐BR system to survival was investigated. First, native H. marismortui and H. salinarum cells were tested in water that had been adjusted to mimic the conditions of Dead Sea water. These archaea were shown to accumulate protons and reduce pH in their periplasmic regions, which disabled further proton transportation functionality in H. salinarum but not in H. marismortui. Then, pH‐dependent photocurrent measurements using purified BR proteins demonstrated that HsBR and HmBRI were functional at pH > 5.0 and that HmBRII was functional at pH > 4.0. Our results indicate that the dual‐HmBR system is composed of two BRs with different optimal functional pH ranges and together they maintain light‐driven proton transport activity under pH > 4.0, which might contribute the survival of H. marismortui under the acidic pH of the Dead Sea.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

An inward proton transport using anabaena sensory rhodopsin

ATP is synthesized by an enzyme that utilizes proton motive force and thus nature creates various proton pumps. The best understood proton pump is bacteriorhodopsin (BR), an outward-directed light-driven proton pump in Halobacterium salinarum. Many archaeal and eubacterial rhodopsins are now known to show similar proton transport activity. Proton pumps must have a specific mechanism to exclude transport in the reverse direction to maintain a proton gradient, and in the case of BR, a highly hydrophobic cytoplasmic domain may constitute such machinery. Although an inward proton pump has neither been created naturally nor artificially, we recently reported that an inward-directed proton transport can be engineered from a bacterial rhodopsin by a single amino acid replacement Anabaena sensory rhodopsin (ASR) is a photochromic sensor in freshwater cyanobacteria, possessing little proton transport activity. When we replace Asp217 at the cytoplasmic domain (distance ～15 Å from the retinal chromophore) to Glu, ASR is converted into an inward proton transport, driven by absorption of a single photon. FTIR spectra clearly show an increased proton affinity for Glu217, which presumably controls the unusual directionality opposite to normal proton pumps.     

Proteorhodopsin: Characterisation of 2D crystals by electron microscopy and solid state NMR

Proteorhodopsin (PR) a recent addition to retinal type 1 protein family, is a bacterial homologue of archaeal bacteriorhodopsin. It was found to high abundance in γ-proteobacteria in the photic zone of the oceans and has been shown to act as a photoactive proton pump. It is therefore involved in the utilisation of light energy for energy production within the cell. Based on data from biodiversity screens, hundreds of variants were discovered worldwide, which are spectrally tuned to the available light at different locations in the sea. Here, we present a characterisation of 2D crystals of the green variant of proteorhodopsin by electron microscopy and solid state NMR. 2D crystal formation with hexagonal protein packing was observed under a very wide range of conditions indicating that PR might be also closely packed under native conditions. A low-resolution 2D projection map reveals a ring-shaped oligomeric assembly of PR. The protein state was analysed by ¹⁵N MAS NMR on lysine, tryptophan and methionine labelled samples. The chemical shift of the protonated Schiff base was almost identical to non-crystalline preparations. All residues could be cross-polarised in non-frozen samples. Lee-Goldberg cross-polarisation has been used to probe protein backbone mobility.     

Orientation and conformation of lipids in crystals of transmembrane proteins

Orientational order parameters and individual dihedral torsion angles are evaluated for phospholipid and glycolipid molecules that are resolved in X-ray structures of integral transmembrane proteins in crystals. The order parameters of the lipid chains and glycerol backbones in protein crystals are characterised by a much wider distribution of orientational order than is found in fluid lipid bilayers and reconstituted lipid–protein membranes. This indicates that the lipids that are resolved in crystals of membrane proteins are mostly not representative of the entire lipid–protein interface. Much of the chain configurational disorder of the membrane-bound lipids in crystals arises from C–C bonds in energetically disallowed skew conformations. This suggests configurational heterogeneity of the lipids at a single binding site: eclipsed conformations occur also in the glycerol backbone torsion angles and the C–C torsion angles of the lipid head groups. Conformations of the lipid glycerol backbone in protein crystals are not restricted to the gauche C1–C2 rotamers found invariably in phospholipid bilayer crystals. Lipid head-group conformations in the protein crystals also do not conform solely to the bent-down conformation, with gauche–gauche configuration of the phosphodiester, that is characteristic of phospholipid bilayer membranes. Stereochemical violations in the protein-bound lipids are evidenced by ester carboxyl groups in non-planar configurations, and even in the cis configuration. Some lipids have the incorrect enantiomeric configuration of the glycerol backbone, and many of the branched methyl groups in the phytanyl chains associated with bacteriorhodopsin have the incorrect S configuration.     

Electrooptical studies on proton-binding and -release of bacteriorhodopsin

Electric field induced pH changes of purple membrane suspensions were investigated in the pH range from 4.1 to 7.6 by measuring the absorbance change of pH indicators. In connection with the photocycle and proton pump ability, three different states of bacteriorhodopsin were used: (1) the native purple bacteriorhodopsin (magnesium and calcium ions are bound, the M intermediate exists in the photocycle and protons are pumped), (2) the cation-depleted blue bacteriorhodopsin (no M intermediate), and (3) the regenerated purple bacteriorhodopsin which is produced either by raising the pH or by adding magnesium ions (the M intermediate exists). In the native purple bacteriorhodopsin there are, at least, two types of proton binding sites: one releases protons and the other takes up protons in the presence of the electric field. On the other hand, blue bacteriorhodopsin and the regenerated purple bacteriorhodopsin (pH increase) show neither proton release nor proton uptake. When magnesium ions are added to the suspensions; the field-induced pH change is observed again. Thus, the stability of proton binding depends strongly on the state of bacteriorhodopsin and differences in proton binding are likely to be related to differences in proton pump activity. Furthermore, it is suggested that the appearance of the M intermediate and proton pumping are not necessarily related.     

Functional reconstitution of a membrane protein in a diacetylenic polymerizable lecithin

It is shown that polymerized diacetylenic lecithins may be used for the functional reconstitution of a membrane protein. Purple membrane patches isolated from Halobacterium halobium and liposomes of the polymerizable diacetylenic lecithin 1,2-bis(10,12 tricosadiynoyl)-sn-glycero-3-phosphocholine were sonicated together to form mixed vesicles highly enriched in the polymerizable lipid. A net inward proton flow on illumination as determined by the change of pH of the external medium demonstrated the stability of the vesicular form in this mixed lipid system as well as vectorial orientation of the bacteriorhodopsin in the bilayer. When bacteriorhodopsin was incorporated in non-polymerizable lipids, irradiation with ultraviolet light resulted in complete loss of function. In the diacetylenic lipids, the loss of function was slower than the increase in polymer concentration. This demonstrates the utility of the diacetylenic lecithin system for study of interactions between membrane proteins and polymerizable lipids, as well as its potential in the development of biosensors based on membrane proteins.     

Influence of the Charge at D85 on the Initial Steps in the Photocycle of Bacteriorhodopsin

Studies have shown that trans-cis isomerization of retinal is the primary photoreaction in the photocycle of the light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum, as well as in the photocycle of the chloride pump halorhodopsin (HR). The transmembrane proteins HR and BR show extensive structural similarities, but differ in the electrostatic surroundings of the retinal chromophore near the protonated Schiff base. Point mutation of BR of the negatively charged aspartate D85 to a threonine T (D85T) in combination with variation of the pH value and anion concentration is used to study the ultrafast photoisomerization of BR and HR for well-defined electrostatic surroundings of the retinal chromophore. Variations of the pH value and salt concentration allow a switch in the isomerization dynamics of the BR mutant D85T between BR-like and HR-like behaviors. At low salt concentrations or a high pH value (pH 8), the mutant D85T shows a biexponential initial reaction similar to that of HR. The combination of high salt concentration and a low pH value (pH 6) leads to a subpopulation of 25% of the mutant D85T whose stationary and dynamic absorption properties are similar to those of native BR. In this sample, the combination of low pH and high salt concentration reestablishes the electrostatic surroundings originally present in native BR, but only a minor fraction of the D85T molecules have the charge located exactly at the position required for the BR-like fast isomerization reaction. The results suggest that the electrostatics in the native BR protein is optimized by evolution. The accurate location of the fixed charge at the aspartate D85 near the Schiff base in BR is essential for the high efficiency of the primary reaction.     

Folding and assembly of proteorhodopsin

Proteorhodopsins (PRs), the recently discovered light-driven proton pumps, play a major role in supplying energy for microbial organisms of oceans. In contrast to PR, rhodopsins found in Archaea and Eukarya are structurally well characterized. Using single-molecule microscopy and spectroscopy, we observed the oligomeric assembly of native PR molecules and detected their folding in the membrane. PR showed unfolding patterns identical with those of bacteriorhodopsin and halorhodopsin, indicating that PR folds similarly to archaeal rhodopsins. Surprisingly, PR predominantly assembles into hexameric oligomers, with a smaller fraction assembling into pentamers. Within these oligomers, PR arranged into radial assemblies. We suggest that this structural assembly of PR may have functional implications.     

Structural role of bacterioruberin in the trimeric structure of archaerhodopsin-2

Archaerhodopsin-2 (aR2), a retinal protein-carotenoid complex found in the claret membrane of Halorubrum sp. aus-2, functions as a light-driven proton pump. In this study, the membrane fusion method was utilized to prepare trigonal P321 crystals (a = b = 98.2 Å, c = 56.2 Å) and hexagonal P6₃ crystals (a = b = 108.8 Å, c = 220.7 Å). The trigonal crystal is made up of stacked membranes in which the aR2 trimers are arranged on a honeycomb lattice. Similar membranous structures are found in the hexagonal crystal, but four membrane layers with different orientations are contained in the unit cell. In these crystals, the carotenoid bacterioruberin [5,32-bis(2-hydroxypropan-2-yl)-2,8,12,16,21,25,29,35-octamethylhexatriaconta-6,8,10,12,14,16,18,20,22,24,26,28,30-tridecaene-2,35-diol] binds to crevices between the subunits of the trimer. Its polyene chain is inclined from the membrane normal by an angle of about 20° and, on the cytoplasmic side, it is surrounded by helices AB and DE of neighbouring subunits. This peculiar binding mode suggests that bacterioruberin plays a striking structural role for the trimerization of aR2. When compared with the aR2 structure in another crystal form containing no bacterioruberin, the proton release channel takes a more closed conformation in the P321 or P6₃ crystal; i.e., the native conformation of protein is stabilized in the trimeric protein-bacterioruberin complex. Interestingly, most residues participating in the trimerization are not conserved in bacteriorhodopsin, a homologous protein capable of forming a trimeric structure in the absence of bacterioruberin. Despite a large alteration in the amino acid sequence, the shape of the intratrimer hydrophobic space filled by lipids is highly conserved between aR2 and bacteriorhodopsin. Since a transmembrane helix facing this space undergoes a large conformational change during the proton pumping cycle, it is feasible that trimerization is an important strategy to capture special lipid components that are relevant to the protein activity.     

Proteorhodopsin: characterisation of 2D crystals by electron microscopy and solid state NMR

Proteorhodopsin (PR) a recent addition to retinal type 1 protein family, is a bacterial homologue of archaeal bacteriorhodopsin. It was found to high abundance in gamma-proteobacteria in the photic zone of the oceans and has been shown to act as a photoactive proton pump. It is therefore involved in the utilisation of light energy for energy production within the cell. Based on data from biodiversity screens, hundreds of variants were discovered worldwide, which are spectrally tuned to the available light at different locations in the sea. Here, we present a characterisation of 2D crystals of the green variant of proteorhodopsin by electron microscopy and solid state NMR. 2D crystal formation with hexagonal protein packing was observed under a very wide range of conditions indicating that PR might be also closely packed under native conditions. A low-resolution 2D projection map reveals a ring-shaped oligomeric assembly of PR. The protein state was analysed by 15N MAS NMR on lysine, tryptophan and methionine labelled samples. The chemical shift of the protonated Schiff base was almost identical to non-crystalline preparations. All residues could be cross-polarised in non-frozen samples. Lee-Goldberg cross-polarisation has been used to probe protein backbone mobility.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

Purple-to-blue transition of bacteriorhodopsin in a neutral lipid environment.

The red shift in the absorption maximum of native purple membrane suspensions caused by deionization is missing in lipid-depleted purple membrane, and the pK of the acid-induced transition is down-shifted to pH approximately 1.4 and has become independent of cation concentration (Szundi, I., and W. Stoeckenius. 1987. Proc. Natl. Acad. Sci. USA. 84:3681-3684). However, the proton pumping function cannot be demonstrated in these membranes. When native acidic lipids of purple membrane are exchanged for egg phosphatidylcholine or digalactosyldiglyceride, bacteriorhodopsin is functionally active in the modified membrane. It shows spectral shifts upon light-dark adaptation, a photocycle with M-intermediate and complex decay kinetics; when reconstituted into vesicles with the same neutral lipids, it pumps protons. Unlike native purple membrane, lipid-substituted modified membranes do not show a shift of the absorption maximum to longer wavelength upon deionization. A partial shift can be induced by titration with HCl; it has a pK near 1.5 and no significant salt dependence. Titration with HNO3 and H2SO4, which causes a complete transition in the lipid-depleted membranes, i.e., it changes their colors from purple to blue, does not cause the complete transition in the lipid-substituted preparations. These results show that the purple color of bacteriorhodopsin is independent of cations and their role in the purple-to-blue transition of native membranes is indirect. The purple and blue colors of bacteriorhodopsin are interpreted as two conformational states of the protein, rather than different protonation states of a counterion to the protonated Schiff base.     

LCP-Tm: An Assay to Measure and Understand Stability of Membrane Proteins in a Membrane Environment

Structural and functional studies of membrane proteins are limited by their poor stability outside the native membrane environment. The development of novel methods to efficiently stabilize membrane proteins immediately after purification is important for biophysical studies, and is likely to be critical for studying the more challenging human targets. Lipidic cubic phase (LCP) provides a suitable stabilizing matrix for studying membrane proteins by spectroscopic and other biophysical techniques, including obtaining highly ordered membrane protein crystals for structural studies. We have developed a robust and accurate assay, LCP-T_m, for measuring the thermal stability of membrane proteins embedded in an LCP matrix. In its two implementations, protein denaturation is followed either by a change in the intrinsic protein fluorescence on ligand release, or by an increase in the fluorescence of a thiol-binding reporter dye that measures exposure of cysteines buried in the native structure. Application of the LCP-T_m assay to an engineered human β₂-adrenergic receptor and bacteriorhodopsin revealed a number of factors that increased protein stability in LCP. This assay has the potential to guide protein engineering efforts and identify stabilizing conditions that may improve the chances of obtaining high-resolution structures of intrinsically unstable membrane proteins.     

His-75 in Proteorhodopsin, a Novel Component in Light-driven Proton Translocation by Primary Pumps

Proteorhodopsins (PRs), photoactive retinylidene membrane proteins ubiquitous in marine eubacteria, exhibit light-driven proton transport activity similar to that of the well studied bacteriorhodopsin from halophilic archaea. However, unlike bacteriorhodopsin, PRs have a single highly conserved histidine located near the photoactive site of the protein. Time-resolved Fourier transform IR difference spectroscopy combined with visible absorption spectroscopy, isotope labeling, and electrical measurements of light-induced charge movements reveal participation of His-75 in the proton translocation mechanism of PR. Substitution of His-75 with Ala or Glu perturbed the structure of the photoactive site and resulted in significantly shifted visible absorption spectra. In contrast, His-75 substitution with a positively charged Arg did not shift the visible absorption spectrum of PR. The mutation to Arg also blocks the light-induced proton transfer from the Schiff base to its counterion Asp-97 during the photocycle and the acid-induced protonation of Asp-97 in the dark state of the protein. Isotope labeling of histidine revealed that His-75 undergoes deprotonation during the photocycle in the proton-pumping (high pH) form of PR, a reaction further supported by results from H75E. Finally, all His-75 mutations greatly affect charge movements within the PR and shift its pH dependence to acidic values. A model of the proteorhodopsin proton transport process is proposed as follows: (i) in the dark state His-75 is positively charged (protonated) over a wide pH range and interacts directly with the Schiff base counterion Asp-97; and (ii) photoisomerization-induced transfer of the Schiff base proton to the Asp-97 counterion disrupts its interaction with His-75 and triggers a histidine deprotonation.A variety of unicellular microorganisms contain primary proton pumps that convert solar energy into a transmembrane electrochemical proton gradient, which is subsequently used by membrane ATP synthases to generate chemical energy. Well known examples of such pumps are the haloarchaeal rhodopsins, photoactive, seven-helix membrane proteins, which include the well studied proton pump bacteriorhodopsin (BR)⁴ from Halobacterium salinarum and BR homologs in other haloarchaea. Recently, a much larger new family of light-driven proton pumps, the proteorhodopsins (PRs), was identified in marine proteobacteria throughout the oceans (1–3). Despite the diverse properties of PRs, including different visible absorption maxima and photocycle rates (4–6), they all share with BR several key conserved residues as well as an all-trans-retinylidene chromophore in their unphotolyzed state, which is covalently bound to transmembrane helix G via a protonated Schiff base linkage.Many of the molecular events that occur in PRs following light activation are similar to those of BR, including an initial ultrafast all-trans→13-cis-retinal isomerization, which triggers a sequence of protein conformational changes, including several intramolecular proton transfer reactions. The two key carboxylate groups involved in proton pumping in helix C of BR are conserved in PRs, and in the first found and most commonly studied PR, the Monterey Bay variant eBAC31A08, also known as green-absorbing proteorhodopsin (GPR), the helix C residues Asp-97 and Glu-108 undergo protonation changes during the photocycle similar to those of the homologous carboxylate residues in BR. Initial FTIR studies on GPR identified the role of Asp-97 as the Schiff base counterion and proton acceptor during Schiff base deprotonation and concomitant M formation and Glu-108 as the proton donor that reprotonates the Schiff base during N formation (7, 8). Studies of other variants indicate these roles of the two carboxylic acid residues are general in the proteorhodopsin family.⁵One major difference between BR and the PRs is the presence of a highly conserved histidine residue at position 75, near the middle of transmembrane helix B in the latter pigments. The His-75 homolog is not present in BR nor thus far found in other microbial rhodopsins (9). The proximity of His-75 to the protein active site and specifically to the Schiff base counterion Asp-97 inferred from the x-ray crystal structure of BR suggests its involvement in spectral tuning of the visible absorption (10) and potentially PR photochemical reactions. Because the pK_a of histidine in solution is close to neutral pH (11), its imidazole group often plays a major role in intramolecular proton transfers in enzymes, including NADPH oxidase (12), alcohol dehydrogenase (13), carbonic anhydrase II (14), and serine proteases (15).In this study we have used a combination of time-resolved FTIR difference spectroscopy, visible absorption spectroscopy, isotope labeling, kinetic charge displacement measurements, and site-directed mutagenesis to study the role of His-75 in GPR. We report evidence that protonated His-75 interacts directly with Asp-97 in the unphotolyzed protein and during the photocycle undergoes a deprotonation in response to the protonation of Asp-97.     

Bacterioopsin, haloopsin, and sensory opsin I of the halobacterial isolate Halobacterium sp. strain SG1: three new members of a growing family.

The genes coding for bacterioopsin, haloopsin, and sensory opsin I of a halobacterial isolate from the Red Sea called Halobacterium sp. strain SG1 have been cloned and sequenced. The deduced protein sequences were aligned to the previously known halobacterial retinal proteins. The addition of these new sequences lowered the number of conserved residues to only 23 amino acids, or 8% of the alignment. Data base searches with two highly conserved peptides as well as with an alignment profile yielded no significant similarity to any other protein, so the halobacterial retinal proteins should be regarded as a distinct protein family. The protein alignment was used to make predictions about the structure of the retinal proteins as well as about the amino acids in contact with retinal proteins. These results were in excellent agreement with the structural model of bacteriorhodopsin of Halobacterium halobium as well as with mutant studies, indicating that (i) structure predictions based on the sequences of a membrane protein family can be quite accurate; (ii) halorhodopsin and sensory rhodopsin I have tertiary structures similar to that of bacteriorhodopsin; (iii) conserved amino acids do not take part in reactions specific for one group of proteins, e.g., proton translocation for bacteriorhodopsins, but have a crucial role in determining the conformation and reactions of the chromophore; and (iv) the general mode of action (light-induced chromophore and protein movements) is the same for all halobacterial retinal proteins, ion pumps as well as sensors.     

The preparation of lipid-depleted bacteriorhodopsin

Bacteriorhodopsin, the protein of the purple membrane of Halobacterium halobium, was freed to the extent of 90–95% from the natural membrane lipids without loss of function. The residual lipid corresponded to less than 1 mol/mol of bacteriorhodopsin. Delipidation was achieved by treatment of the purple membrane with a mixture of the detergent dimethyldodecylamine oxide and sodium chloride. The detergent was removed by dialysis or by sucrose density gradient centrifugation. Analysis of the lipids removed and those still bound to bacteriorhodopsin was facilitated by the use of purple membrane preparations labelled with ³⁵S, ³²P, or ¹⁴C. The composition of the residual lipids associated with bacteriorhodopsin was similar to that of the total lipid in the purple membrane.     

Elimination of proton donor strongly affects directionality and efficiency of proton transport in ESR,a light-driven proton pump from Exiguobacterium sibiricum

ESR from Exiguobacterium sibiricum is a retinal protein which functions as a proton pump. Unusual feature of ESR is that a lysine residue is present at a site for the internal proton donor, which in other proton pumps is a carboxylic residue. Replacement of Lys96 with alanine slows reprotonation of the Schiff base by two orders of magnitude, indicating that Lys96 and interacting water molecules function as internal proton donor to the Schiff base. In this work we examined time resolved generation of light-induced electric potential ΔΨ by the K96A mutant reconstituted into proteoliposomes. We found that the ΔΨ component, which accompanied reprotonation of the Schiff base in wild type ESR, was not only slowed but also decreased greatly in the mutant, and negative phase appeared at high pH. This indicates a higher probability of back reactions in ESR than in bacteriorhodopsin since no negative components have been observed in homologous mutants of BR, D96N and D96A. The higher rate of back reactions in ESR is probably caused by different arrangement of the proton acceptor site compared to that in BR and different sequence of proton release and uptake. Addition of sodium azide, which substitutes for the internal proton donor, restores both the rate and amplitude of the ΔΨ components related to the Schiff base reprotonation in the K96A mutant. This indicates that overall proton transport results from competition of forward and reverse reactions, and emphasizes the importance of internal donor for high efficiency and directionality of H⁺ transfer.     

Bacterial chimeras and reversible phosphorylation: the work of Walther Stoeckenius

In 1971, Walther Stoeckenius discovered that Halobacterium halobium contains a purple pigment that is chemically similar to rhodopsin and works as a light-driven proton pump. This discovery set Stoeckenius on a research path centered on bacteriorhodopsin, which included the creation of a bovine-soybean-halobacteria chimera that was able to produce ATP when exposed to light and the discovery of a class of proteins that are phosphorylated in a light-dependent manner.Reconstitution of Purple Membrane Vesicles Catalyzing Light-driven Proton Uptake and Adenosine Triphosphate Formation (Racker, E., and Stoeckenius, W. (1974) J. Biol. Chem. 249, 662–663)Light-regulated Retinal-dependent Reversible Phosphorylation of Halobacterium Proteins (Spudich, J. L., and Stoeckenius, W. (1980) J. Biol. Chem. 255, 5501–5503)Walther Stoeckenius was born in 1921 in Giessen, Germany. He earned an M.D. degree from the University of Hamburg in 1950, after which he spent 18 months doing clinical work as an intern. In 1952, he began postdoctoral work at the Institute for Tropical Medicine in Hamburg, using electron microscopy to study the development of pox viruses. Two years later, he joined the Department of Pathology at the University of Hamburg as an assistant professor and became Docent for Pathology in 1958. At Hamburg, Stoeckenius continued to use electron microscopy to explore the fine structure of cells and the lipid membrane.In 1959, Stoeckenius left Germany to become a research associate in Keith Porter''s laboratory at Rockefeller University. After a few months, he became an assistant professor at Rockefeller, remaining there for 8 years and eventually becoming an associate professor. He continued to work on membrane structure, studying Halobacterium halobium, until he accepted a professorship at the University of California, San Francisco in 1967.In San Francisco, Stoeckenius focused more on biochemical techniques rather than electron microscopy. In collaboration with Dieter Oesterhelt, he discovered that H. halobium contains a purple pigment (bacteriorhodopsin) that is chemically similar to rhodopsin (1) and plays an important role in light energy storage in halobacteria, working as a light-driven proton pump (2).This discovery led to a collaboration with Journal of Biological Chemistry (JBC) Classic author Efraim Racker (3) in which Stoeckenius and Racker created a thoroughly unnatural vesicle. As reported in the first JBC Classic reprinted here, they used sonication to recombine membrane lipids from soybeans, bacteriorhodopsin from halobacteria, and ATPase from beef mitochondria. The resulting artificial vesicles were able to produce ATP when exposed to light. The chimeric vesicles also formed a simple model system for a biological proton pump capable of generating ATP from ADP and P_i.Stoeckenius continued to study bacteriorhodopsin and its light-driven proton uptake in bacteria. As reported in the second JBC Classic reprinted here, he discovered that phosphorylation is regulated by light absorbed by bacteriorhodopsin (4). Using [³²P]orthophosphate pulse labeling, Stoeckenius and John Spudich identified a class of phosphoproteins in H. halobium. Exposing labeled whole cells to light resulted in rapid dephosphorylation of two of the proteins, which were rapidly rephosphorylated upon darkening of the cells. The light sensitivity of the proteins was responsive to the presence of retinal, indicating that the dephosphorylation depended on rhodopsin-like (retinal-containing) photoreceptors.Stoeckenius currently is Professor Emeritus in the Department of Biochemistry and Biophysics and the Cardiovascular Research Institute at the University of California, San Francisco. He was elected to the National Academy of Sciences in 1978.     

Insights into archaeal chaperone machinery: a network-based approach

Molecular chaperones are a diverse group of proteins that ensure proteome integrity by helping the proteins fold correctly and maintain their native state, thus preventing their misfolding and subsequent aggregation. The chaperone machinery of archaeal organisms has been thought to closely resemble that found in humans, at least in terms of constituent players. Very few studies have been ventured into system-level analysis of chaperones and their functioning in archaeal cells. In this study, we attempted such an analysis of chaperone-assisted protein folding in archaeal organisms through network approach using Picrophilus torridus as model system. The study revealed that DnaK protein of Hsp70 system acts as hub in protein-protein interaction network. However, DnaK protein was present only in a subset of archaeal organisms and absent from many archaea, especially members of Crenarchaeota phylum. Therefore, a similar network was created for another archaeal organism, Sulfolobus solfataricus, a member of Crenarchaeota. The chaperone network of S. solfataricus suggested that thermosomes played an integral part of hub proteins in archaeal organisms, where DnaK was absent. We further compared the chaperone network of archaea with that found in eukaryotic systems, by creating a similar network for Homo sapiens. In the human chaperone network, the UBC protein, a part of ubiquitination system, was the most important module, and interestingly, this system is known to be absent in archaeal organisms. Comprehensive comparison of these networks leads to several interesting conclusions regarding similarities and differences within archaeal chaperone machinery in comparison to humans.