Biomedical subjects

Peter Dimroth

Publications and source records attributed to Peter Dimroth.

At least 19 recordsLinked to original sources

PubMed2003

Observing membrane protein diffusion at subnanometer resolution.

Single sodium-driven rotors from a bacterial ATP synthase were embedded into a lipid membrane and observed in buffer solution at subnanometer resolution using atomic force microscopy (AFM). Time-lapse AFM topographs show the movement of single proteins within the membrane. Subsequent analysis of their individual trajectories, in consideration of the environment surrounding the moving protein, allow principal modes of the protein motion to be distinguished. Within one trajectory, individual proteins can undergo movements assigned to free as well as to obstacled diffusion. The diffusion constants of these two modes of motion are considerably different. Without the structural information about the membrane environment restricting the moving proteins, it would not be possible to reveal insight into these mechanisms. The high-resolution AFM topographs suggest that, in future studies, such data revealed under various physiological conditions will provide novel insights into molecular mechanisms that drive membrane protein assembly and supply excellent boundary conditions to model protein-protein arrangements.

Diffusion↗

PubMed2003

Sodium ion cycling mediates energy coupling between complex I and ATP synthase.

We show here sodium ion cycling between complex I from Klebsiella pneumoniae and the F(1)F(0) ATP synthase from Ilyobacter tartaricus in a reconstituted proteoliposome system. In the course of NADH oxidation by complex I, an electrochemical sodium ion gradient was established and served as a driving force for the synthesis of ATP from ADP and phosphate. In the opposite direction, the deltamu(Na(+)) generated by ATP hydrolysis could be coupled to NADH formation by reversed electron transfer from ubiquinol to NAD. For reverse electron transfer, a transmembrane voltage larger than 30 mV was obligatory. No NADH-driven proton transport into the lumen of proteoliposomes was detected. We conclude that Na(+) is used as the exclusive coupling ion by the enterobacterial complex I.

Adenosine Triphosphate↗

PubMed2003

Evidence for structural integrity in the undecameric c-rings isolated from sodium ATP synthases.

The Na(+)-translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contain undecameric c subunit rings of unusual stability. These c(11) rings have been isolated from both ATP synthases and crystallized in two dimensions. Cryo-transmission electron microscopy projection maps of the c-rings from both organisms were identical at 7A resolution. Different crystal contacts were induced after treatment of the crystals with dicyclohexylcarbodiimide (DCCD), which is consistent with the binding of the inhibitor to glutamate 65 in the C-terminal helix on the outside of the ring. The c subunits of the isolated c(11) ring of I.tartaricus were modified specifically by incubation with DCCD with kinetics that were indistinguishable from those of the F(1)F(o) holoenzyme. The reaction rate increased with decreasing pH but was lower in the presence of Na(+). From the pH profile of the second-order rate constants, the pK of glutamate 65 was deduced to be 6.6 or 6.2 in the absence or presence of 0.5mM NaCl, respectively. These pK values are identical with those determined for the F(1)F(o) complex. The results indicate that the isolated c-ring retains its native structure, and that the glutamate 65, including binding sites near the middle of the membrane, are accessible to Na(+) from the cytoplasm through access channels within the c-ring itself.

Bacterial Proteins↗

PubMed2003

Charge displacements during ATP-hydrolysis and synthesis of the Na+-transporting FoF1-ATPase of Ilyobacter tartaricus.

Transient electrical currents generated by the Na(+)-transporting F(o)F(1)-ATPase of Ilyobacter tartaricus were observed in the hydrolytic and synthetic mode of the enzyme. Two techniques were applied: a photochemical ATP concentration jump on a planar lipid membrane and a rapid solution exchange on a solid supported membrane. We have identified an electrogenic reaction in the reaction cycle of the F(o)F(1)-ATPase that is related to the translocation of the cation through the membrane bound F(o) subcomplex of the ATPase. In addition, we have determined rate constants for the process: For ATP hydrolysis this reaction has a rate constant of 15-30 s(-1) if H(+) is transported and 30-60 s(-1) if Na(+) is transported. For ATP synthesis the rate constant is 50-70 s(-1).

Adenosine Diphosphate↗

PubMed2003

The citrate carrier CitS probed by single-molecule fluorescence spectroscopy.

A prominent region of the Na(+)-dependent citrate carrier (CitS) from Klebsiella pneumoniae is the highly conserved loop X-XI, which contains a putative citrate binding site. To monitor potential conformational changes within this region by single-molecule fluorescence spectroscopy, the target cysteines C398 and C414 of the single-Cys mutants (CitS-sC398, CitS-sC414) were selectively labeled with the thiol-reactive fluorophores AlexaFluor 546/568 C(5) maleimide (AF(546), AF(568)). While both single-cysteine mutants were catalytically active citrate carriers, labeling with the fluorophore was only tolerated at C398. Upon citrate addition to the functional protein fluorophore conjugate CitS-sC398-AF(546), complete fluorescence quenching of the majority of molecules was observed, indicating a citrate-induced conformational change of the fluorophore-containing domain of CitS. This quenching was specific for the physiological substrate citrate and therefore most likely reflecting a conformational change in the citrate transport mechanism. Single-molecule studies with dual-labeled CitS-sC398-AF(546/568) and dual-color detection provided strong evidence for a homodimeric association of CitS.

Bacterial Proteins↗

PubMed2003

Purification and biochemical characterization of the F1Fo-ATP synthase from thermoalkaliphilic Bacillus sp. strain TA2.A1.

We describe here purification and biochemical characterization of the F(1)F(o)-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F(1)F(o)-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F(1) subunits alpha, beta, gamma, delta, and epsilon and F(o) subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F(1)F(o) holoenzyme or the isolated F(1) moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F(1). After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Deltapsi). A transmembrane proton gradient or sodium ion gradient in the absence of Deltapsi was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na(+)/H(+) antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na(+) ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N'-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.

Adenosine Triphosphate↗

PubMed2003

Bioenergetic properties of the thermoalkaliphilic Bacillus sp. strain TA2.A1.

The thermoalkaliphilic Bacillus sp. strain TA2.A1 was able to grow in pH-controlled batch culture containing a nonfermentable growth substrate from pH 7.5 to 10.0 with no significant change in its specific growth rate, demonstrating that this bacterium is a facultative alkaliphile. Growth at pH 10.0 was sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that a proton motive force (Deltap) generated via aerobic respiration was an obligate requirement for growth of strain TA2.A1. Strain TA2.A1 exhibited intracellular pH homeostasis as the external pH increased from 7.5 to 10.0; however, the maximum DeltapH generated over this pH range was only 1.1 units at an external pH of 9.5. The membrane potential (Deltapsi) was maintained between -114 mV and -150 mV, and little significant change was observed over the pH range for growth. In contrast, the Deltap declined from -164 mV at pH 7.5 to approximately -78 mV at pH 10.0. An inwardly directed sodium motive force (DeltapNa(+)) of -100 mV at pH 10.0 indicated that cellular processes (i.e., solute transport) dependent on a sodium gradient would not be affected by the adverse Deltap. The phosphorylation potential of strain TA2.A1 was maintained between -300 mV and -418 mV, and the calculated H(+)/ATP stoichiometry of the ATP synthase increased from 2.0 at pH 7.5 to 5.7 at pH 10.0. Based on these data, vigorous growth of strain TA2.A1 correlated well with the DeltapNa(+), phosphorylation potential, and the ATP/ADP ratio, but not with Deltap. This communication represents the first report on the bioenergetics of an extremely thermoalkaliphilic aerobic bacterium.

Adenosine Triphosphate↗

PubMed2002

Intersubunit bridging by Na+ ions as a rationale for the unusual stability of the c-rings of Na+-translocating F1F0 ATP synthases.

The oligomeric c-rings of Na+-translocating F1F0 ATP synthases exhibit unusual stability, resisting even boiling in SDS. Here, we show that the molecular basis for this remarkable property is intersubunit crossbridging by Na+ or Li+ ions. The heat stability of c11 was dependent on the presence of Na+ or Li+ ions. For equal stability, 10 times higher Li+ than Na+ concentrations were required, reflecting the 10 times lower binding affinity for Li+ than for Na+. In a recent structural model of c11, the Na+ or Li+ binding ligands are located on neighboring c-subunits, which thus become crossbridged by the binding of either alkali ion with a concomitant increase in the stability of the ring. Site-directed mutagenesis strengthens the essential role of glutamate 65 in the crossbridging of the subunits and also corroborates the proposed stabilizing effect of an ion bridge including aspartate 2.

Bacterial Proteins↗

PubMed2002

Molecular mechanism of the ATP synthase's F(o) motor probed by mutational analyses of subunit a.

The most prominent residue of subunit a of the F(1)F(o) ATP synthase is a universally conserved arginine (aR227 in Propionigenium modestum), which was reported to permit no substitution with retention of ATP synthesis or H(+)-coupled ATP hydrolysis activity. We show here that ATP synthases with R227K or R227H mutations in the P.modestum a subunit catalyse ATP-driven Na(+) transport above or below pH 8.0, respectively. Reconstituted F(o) with either mutation catalysed 22Na(+)(out)/Na(+)(in) exchange with similar pH profiles as found in ATP-driven Na(+) transport. ATP synthase with an aR227A substitution catalysed Na(+)-dependent ATP hydrolysis, which was completely inhibited by dicyclohexylcarbodiimide, but not coupled to Na(+) transport. This suggests that in the mutant the dissociation of Na(+) becomes more difficult and that the alkali ions remain therefore permanently bound to the c subunit sites. The reconstituted mutant enzyme was also able to synthesise ATP in the presence of a membrane potential, which stopped at elevated external Na(+) concentrations. These observations reinforce the importance of aR227 to facilitate the dissociation of Na(+) from approaching rotor sites. This task of aR227 was corroborated by other results with the aR227A mutant: (i) after reconstitution into liposomes, F(o) with the aR227A mutation did not catalyse 22Na(+)(out)/Na(+)(in) exchange at high internal sodium concentrations, and (ii) at a constant (Delta)pNa(+), 22Na(+) uptake was inhibited at elevated internal Na(+) concentrations. Hence, in mutant aR227A, sodium ions can only dissociate from their rotor sites into a reservoir of low sodium ion concentration, whereas in the wild-type the positively charged aR227 allows the dissociation of Na(+) even into compartments of high Na(+) concentration.

Adenosine Triphosphate↗

PubMed2002

Coupled rotation within single F0F1 enzyme complexes during ATP synthesis or hydrolysis.

F0F1 ATP synthases are the smallest rotary motors in nature and work as ATP factories in bacteria, plants and animals. Here we report on the first observation of intersubunit rotation in fully coupled single F0F1 molecules during ATP synthesis or hydrolysis. We investigate the Na+-translocating ATP synthase of Propionigenium modestum specifically labeled by a single fluorophore at one c subunit using polarization-resolved confocal microscopy. Rotation during ATP synthesis was observed with the immobilized enzyme reconstituted into proteoliposomes after applying a diffusion potential, but not with a Na+ concentration gradient alone. During ATP hydrolysis, stepwise rotation of the labeled c subunit was found in the presence of 2 mM NaCl, but not without the addition of Na+ ions. Moreover, upon the incubation with the F0-specific inhibitor dicyclohexylcarbodiimide the rotation was severely inhibited.

Adenosine Triphosphate↗

PubMed2002

Molecular architecture of the undecameric rotor of a bacterial Na+-ATP synthase.

The sodium ion-translocating F(1)F(0) ATP synthase from the bacterium Ilyobacter tartaricus contains a remarkably stable rotor ring composed of 11 c subunits. The rotor ring was isolated, crystallised in two dimensions and analysed by electron cryo-microscopy. Here, we present an alpha-carbon model of the c-subunit ring. Each monomeric c subunit of 89 amino acid residues folds into a helical hairpin consisting of two membrane-spanning helices and a cytoplasmic loop. The 11 N-terminal helices are closely spaced within an inner ring surrounding a cavity of approximately 17A (1.7 nm). The tight helix packing leaves no space for side-chains and is accounted for by a highly conserved motif of four glycine residues in the inner, N-terminal helix. Each inner helix is connected by a clearly visible loop to an outer C-terminal helix. The outer helix has a kink near the position of the ion-binding site residue Glu65 in the centre of the membrane and another kink near the C terminus. Two helices from the outer ring and one from the inner ring form the ion-binding site in the middle of the membrane and a potential access channel from the binding site to the cytoplasmic surface. Three possible inter-subunit ion-bridges are likely to account for the remarkable temperature stability of I.tartaricus c-rings compared to those of other organisms.

Amino Acid Sequence↗

PubMed2002

The respiratory complex I (NDH I) from Klebsiella pneumoniae, a sodium pump.

The electrogenic NADH:Q oxidoreductase from the enterobacterium Klebsiella pneumoniae transports Na(+) ions. The complex was purified with an increase of the specific Na(+) transport activity from 0.2 micromol min(-1) mg(-1) in native membrane vesicles to 4.7 micromol min(-1) mg(-1) in reconstituted enzyme specimens. The subunit pattern resembled that of complex I from Escherichia coli, and two prominent polypeptides were identified as the NuoF and NuoG subunits of complex I. During purification the typical cofactors of complex I were enriched to yield approximately 17 nmol mg(-1) iron, 24 nmol mg(-1) acid-labile sulfide, and 0.79 nmol mg(-1) FMN in the purified sample. The enzyme contained approximately 1.2 nmol mg(-1) Q6 and 1.5 nmol mg(-1) Q8. The reduction of ubiquinone by NADH was Na(+)-dependent, which indicates coupling of the chemical and the vectorial reaction of the pump. The Na(+) activation profile corresponded to the Hill equation with a Hill coefficient K(H)(Na(+)) = 1.96 and with a half-maximal saturation at 0.33 mm Na(+). The reconstituted complex I from Klebsiella pneumoniae catalyzed deamino-NADH oxidation, Q1 reduction, and Na(+) translocation with specific activities of 2.6 units mg(-1), 2.4 units mg(-1), and 4.7 units mg(-1), respectively, which indicate a Na(+)/electron stoichiometry of one.

Ion Transport↗

PubMed2002

Characterization of the citrate/acetate antiporter CitW of Klebsiella pneumoniae.

The genome of Klebsiella pneumoniae contains at least three different genes encoding citrate transporters. Recently, a third and hitherto unknown gene encoding a citrate transport system ( citW) was identified. Escherichia coli transformed with a plasmid expressing citW was able to grow on citrate as sole carbon and energy source, identifying CitW as a citrate carrier. In this report, we provide evidence that further specifies CitW as a Na(+)-independent citrate/citrate and citrate/acetate exchanger. Kinetic analysis of citrate uptake at different pH values identified Hcitrate(2-) as the transported citrate species, with a K(m) of 25 microM. Since citW is expressed under anoxic conditions and acetate is the main end-product of citrate fermentation in K. pneumoniae, citrate/acetate exchange might be its in vivo function. Sequence similarity searches identified CitW (454 amino acids, 48.15 kDa) as a member of the 2-hydroxycarboxylate transporter family (TC 2.A.24). The substrate specificity seems to partially contradict this phylogenetic classification, but appears logical with respect to the putative functional role of CitW in the citrate fermentation pathway of K. pneumoniae.

Acetates↗

PubMed2002

Subunit gamma of the oxaloacetate decarboxylase Na(+) pump: interaction with other subunits/domains of the complex and binding site for the Zn(2+) metal ion.

The oxaloacetate decarboxylase Na(+) pump of Klebsiella pneumoniae is an enzyme complex composed of the peripheral alpha subunit and the two integral membrane-bound subunits beta and gamma. The alpha subunit consists of the N-terminal carboxyltransferase domain and the C-terminal biotin domain, which are connected by a flexible proline/alanine-rich linker peptide. To probe interactions between the two domains of the alpha subunit and between alpha-subunit domains and the gamma subunit, the relevant polypeptides were synthesized in Escherichia coli and subjected to copurification studies. The two alpha-subunit domains had no distinct affinity toward each other and could, therefore, not be purified as a unit on avidin-sepharose. The two domains reacted together catalytically, however, performing the carboxyl transfer from oxaloacetate to protein-bound biotin. This reaction was enhanced up to 6-fold in the presence of the Zn(2+)-containing gamma subunit. On the basis of copurification with different tagged proteins, the C-terminal biotin domain but not the N-terminal carboxyltransferase domain of the alpha subunit formed a strong complex with the gamma subunit. Upon the mutation of gamma H78 to alanine, the binding affinity to subunit alpha was lost, indicating that this amino acid may be essential for formation of the oxaloacetate decarboxylase enzyme complex. The binding residues for the Zn(2+) metal ion were identified by site-directed and deletion mutagenesis. In the gamma D62A or gamma H77A mutant, the Zn(2+) content of the decarboxylase decreased to 35% or 10% of the wild-type enzyme, respectively. Less than 5% of the Zn(2+) present in the wild-type enzyme was found if the two C-terminal gamma-subunit residues H82 and P83 were deleted. Corresponding with the reduced Zn(2+) contents in these mutants, the oxaloacetate decarboxylase activities were diminished. These results indicate that aspartate 62, histidine 77, and histidine 82 of the gamma subunit are ligands for the catalytically important Zn(2+) metal ion.

Base Sequence↗

PubMed2002

Inactivation of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus by reactive oxygen species.

The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) from Vibrio alginolyticus was inactivated by reactive oxygen species. Highest Na+-NQR activity was observed in anaerobically prepared membranes that exhibited 1:1 coupling of NADH oxidation and Q reduction activities (1.6 U x mg(-1)). Optical and EPR spectroscopy documented the presence of b-type cytochromes, a [2Fe-2S] cluster and an organic radical signal in anaerobically prepared membranes from V. alginolyticus. It is shown that the [2Fe-2S] cluster previously assigned to the Na+-NQR originates from the succinate dehydrogenase or the related enzyme fumarate reductase.

Bacterial Proteins↗

PubMed2002

NMR investigations of subunit c of the ATP synthase from Propionigenium modestum in chloroform/methanol/water (4 : 4 : 1).

The subunit c from the ATP synthase of Propionigenium modestum was studied by NMR in chloroform/methanol/water (4 : 4 : 1). In this solvent, subunit c consists of two helical segments, comprised of residues L5 to I26 and G29 to N82, respectively. On comparing the secondary structure of subunit c from P. modestum in the organic solvent mixture with that in dodecylsulfate micelles several deviations became apparent: in the organic solvent, the interruption of the alpha helical structure within the conserved GXGXGXGX motif was shortened from five to two residues, the prominent interruption of the alpha helical structure in the cystoplasmic loop region was not apparent, and neither was there a break in the alpha helix after the sodium ion-binding Glu65 residue. The folding of subunit c of P. modestum in the organic solvent also deviated from that of Escherichia coli in the same environment, the most important difference being that subunit c of P. modestum did not adopt a stable hairpin structure like subunit c of E. coli.

Amino Acid Sequence↗

PubMed2002

Reconstitution of Fo of the sodium ion translocating ATP synthase of Propionigenium modestum from its heterologously expressed and purified subunits.

The atpB and atpF genes of Propionigenium modestum were cloned as His-tag fusion constructs and expressed in Escherichia coli. Both recombinant subunits a and b were purified via Ni(2+) chelate affinity chromatography. A functionally active Fo complex was reassembled in vitro from subunits a, b and c, and incorporated into liposomes. The F(o) liposomes catalysed (22)Na(+) uptake in response to an inside negative potassium diffusion potential, and the uptake was prevented by modification of the c subunits with N,N'-dicyclohexylcarbodiimide (DCCD). In the absence of a membrane potential the Fo complexes catalysed (22)Na(+)(out)/Na(+)(in)-exchange. After F(1) addition the F(1)F(o) complex was formed and the holoenzyme catalysed ATP synthesis, ATP dependent Na(+) pumping, and ATP hydrolysis, which was inhibited by DCCD. Functional F(o) hybrids were reconstituted with recombinant subunits a and b from P. modestum and c(11) from Ilyobacter tartaricus. These Fo hybrids had Na(+) translocation activities that were not distinguishable from that of P. modestum F(o).

Cloning, Molecular↗

PubMed2002

Role of conserved residues within helices IV and VIII of the oxaloacetate decarboxylase beta subunit in the energy coupling mechanism of the Na+ pump.

The membrane-bound beta subunit of the oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae catalyzes the decarboxylation of enzyme-bound biotin. This event is coupled to the transport of 2 Na+ ions into the periplasm and consumes a periplasmically derived proton. The connecting fragment IIIa and transmembrane helices IV and VIII of the beta subunit are highly conserved, harboring residues D203, Y229, N373, G377, S382, and R389 that play a profound role in catalysis. We report here detailed kinetic analyses of the wild-type enzyme and the beta subunit mutants N373D, N373L, S382A, S382D, S382T, R389A, and R389D. In these studies, pH profiles, Na+ binding affinities, Hill coefficients, Vmax values and inhibition by Na+ was determined. A prominent result is the complete lack of oxaloacetate decarboxylase activity of the S382A mutant at Na+ concentrations up to 20 mm and recovery of significant activities at elevated Na+ concentrations (KNa approximately 400 mm at pH 6.0), where the wild-type enzyme is almost completely inhibited. These results indicate impaired Na+ binding to the S382 including site in the S382A mutant. Oxaloacetate decarboxylation by the S382A mutant at high Na+ concentrations is uncoupled from the vectorial events of Na+ or H+ translocation across the membrane. Based on all data with the mutant enzymes we propose a coupling mechanism, which includes Na+ binding to center I contributed by D203 (region IIIa) and N373 (helix VIII) and center II contributed by Y229 (helix IV) and S382 (helix VIII). These centers are exposed to the cytoplasmic surface in the carboxybiotin-bound state of the beta subunit and become exposed to the periplasmic surface after decarboxylation of this compound. During the countertransport of 2 Na+ and 1 H+ Y229 of center II switches between the protonated and deprotonated Na+-bound state.

Amino Acid Substitution↗