45 research outputs found
Proton-Translocating Transhydrogenase from E.COLI. Structure and Function of the Membrane Domain and the NADP(H)- Binding Domain
No full textCoupling through the "hinge" region: roles of individual amino acids in helix 13 and 14 of domain II in E-coli transhydrogenase
No full textShip-Generated Waves and Induced Turbidity in the Gota alv River in Sweden
No full textShip-generated waves were investigated in the Gota alv river, which is a major waterway on the Swedish west coast between the sea and Lake Vanern. Ships with a typical size of 85x15x5 m (lengthxwidthxdraft) travel at speeds between 5 and 10 knots, generating waves that cause sediment transport and erosion along the river bed and banks. Field measurements of the wave properties and turbidity were carried out during 17 ship passages, and comparisons were made with the most commonly used formulas for predicting ship waves. The formula proposed by the Permanent International Association of Navigation Congresses yielded the overall best agreement for the divergent (secondary) waves, whereas the drawdown (primary wave) could best be estimated from the vessel sinkage. The maximum recorded turbidity was mainly a function of the drawdown, and it could be well predicted from the parameterized bed shear stress. In conclusion, ship waves often induce bed and bank erosion in restricted waterways and, although simplistic formulas involve significant uncertainties, they are still useful tools for predictions. However, more studies are needed to determine the influence of a limited river cross section on the wave generation and the relationship between ship waves and sediment transport
Roles of Individual Amino Acids in Helix 14 of the Membrane Domain of Proton-Translocating Transhydrogenase from Escherichia coli As Deduced from Cysteine Mutagenesis
No full textProton-translocating nicotinamide nucleotide transhydrogenase is a membrane-bound protein composed of three domains: the hydrophilic NAD(H)-binding domain, the hydrophilic NADP(H)-binding domain, and the hydrophobic membrane domain. The latter harbors the proton channel. In Escherichia coli transhydrogenase, the membrane domain is composed of 13 transmembrane helices, of which especially helices 13 and 14 contain conserved residues. To characterize the roles of the individual residues Leu240 to Ser260 in helix 14, these were mutated as single mutants to cysteines in the cysteine-free background, and in the case of Gly245, Gly249, and Gly252, also to leucines. In addition to the residues forming the helix, residues Asn238 and Asp239 were also mutated. Except for I242C, all mutants were normally expressed, purified, and characterized with respect to, e.g., catalytic activities and proton pumping. The results show that mutation of the conserved glycines Gly245, Gly249, and Gly252, located on the same face of the helix, led to a general inhibition of all activities, especially in the case of Gly252, suggesting a role of these glycines in helix-helix interactions. In contrast, mutation of the conserved serines Ser250, Ser251, and Ser256 led to enhanced activities of all reactions, including the cyclic reaction which was mediated by bound NADP(H). Mutation of the remaining residues resulted in intermediate inhibitory effects. The results strongly support an important regulatory role of the membrane domain on the NADP(H)-binding site
Properties of the apo-form of the NADP(H)-binding domain III of proton-pumping Escherichia coli transhydrogenase: implications for the reaction mechanism of the intact enzyme
No full textAbstractProton-translocating nicotinamide nucleotide transhydrogenases contain an NAD(H)-binding domain (dI), an NADP(H)-binding domain (dIII) and a membrane domain (dII) with the proton channel. Separately expressed and isolated dIII contains tightly bound NADP(H), predominantly in the oxidized form, possibly representing a so-called “occluded” intermediary state of the reaction cycle of the intact enzyme. Despite a Kd in the micromolar to nanomolar range, this NADP(H) exchanges significantly with the bulk medium. Dissociated NADP+ is thus accessible to added enzymes, such as NADP-isocitrate dehydrogenase, and can be reduced to NADPH. In the present investigation, dissociated NADP(H) was digested with alkaline phosphatase, removing the 2′-phosphate and generating NAD(H). Surprisingly, in the presence of dI, the resulting NADP(H)-free dIII catalyzed a rapid reduction of 3-acetylpyridine-NAD+ by NADH, indicating that 3-acetylpyridine-NAD+ and/or NADH interacts unspecifically with the NADP(H)-binding site. The corresponding reaction in the intact enzyme is not associated with proton pumping. It is concluded that there is a 2′-phosphate-binding region in dIII that controls tight binding of NADP(H) to dIII, which is not a required for fast hydride transfer. It is likely that this region is the Lys424–Arg425–Ser426 sequence and loops D and E. Further, in the intact enzyme, it is proposed that the same region/loops may be involved in the regulation of NADP(H) binding by an electrochemical proton gradent
Ship-Generated Waves and Induced Turbidity in the Gota alv River in Sweden
No full textShip-generated waves were investigated in the Gota alv river, which is a major waterway on the Swedish west coast between the sea and Lake Vanern. Ships with a typical size of 85x15x5 m (lengthxwidthxdraft) travel at speeds between 5 and 10 knots, generating waves that cause sediment transport and erosion along the river bed and banks. Field measurements of the wave properties and turbidity were carried out during 17 ship passages, and comparisons were made with the most commonly used formulas for predicting ship waves. The formula proposed by the Permanent International Association of Navigation Congresses yielded the overall best agreement for the divergent (secondary) waves, whereas the drawdown (primary wave) could best be estimated from the vessel sinkage. The maximum recorded turbidity was mainly a function of the drawdown, and it could be well predicted from the parameterized bed shear stress. In conclusion, ship waves often induce bed and bank erosion in restricted waterways and, although simplistic formulas involve significant uncertainties, they are still useful tools for predictions. However, more studies are needed to determine the influence of a limited river cross section on the wave generation and the relationship between ship waves and sediment transport
Cross-linking of transmembrane helices in proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli: implications for the structure and function of the membrane domain
No full textAbstractProton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α and a β subunit of 54 and 49 kDa, respectively, and is made up of three domains. Domain I (dI) and III (dIII) are hydrophilic and contain the NAD(H)- and NADP(H)-binding sites, respectively, whereas the hydrophobic domain II (dII) contains 13 transmembrane α-helices and harbours the proton channel. Using a cysteine-free transhydrogenase, the organization of dII and helix–helix distances were investigated by the introduction of one or two cysteines in helix–helix loops on the periplasmic side. Mutants were subsequently cross-linked in the absence and presence of diamide and the bifunctional maleimide cross-linker o-PDM (6 Å), and visualized by SDS-PAGE.In the α2β2 tetramer, αβ cross-links were obtained with the αG476C-βS2C, αG476C-βT54C and αG476C-βS183C double mutants. Significant αα cross-links were obtained with the αG476C single mutant in the loop connecting helix 3 and 4, whereas ββ cross-links were obtained with the βS2C, βT54C and βS183C single mutants in the beginning of helix 6, the loop between helix 7 and 8 and the loop connecting helix 11 and 12, respectively. In a model based on 13 mutants, the interface between the α and β subunits in the dimer is lined along an axis formed by helices 3 and 4 from the α subunit and helices 6, 7 and 8 from the β subunit. In addition, helices 2 and 4 in the α subunit together with helices 6 and 12 in the β subunit interact with their counterparts in the α2β2 tetramer. Each β subunit in the α2β2 tetramer was concluded to contain a proton channel composed of the highly conserved helices 9, 10, 13 and 14
The organization of the membrane domain and its interaction with the NADP(H)-binding site in proton-translocating transhydrogenase from E. coli
No full textAbstractProton-translocating nicotinamide nucleotide transhydrogenase is a conformationally driven pump which catalyzes the reversibel reduction of NADP+ by NADH. Transhydrogenases contain three domains, i.e., the hydrophilic NAD(H)-binding domain I and the NADP(H)-binding domain III, and the hydrophobic domain II containing the proton channel. Domains I and III have been separately expressed and characterized structurally by, e.g. X-ray crystallography and NMR. These domains catalyze transhydrogenation in the absence of domain II. However, due to the absence of the latter domain, the reactions catalyzed by domains I and III differ significantly from those catalyzed by the intact enzyme. Mutagenesis of residues in domain II markedly affects the activity of the intact enzyme. In order to resolve the structure–function relationships of the intact enzyme, and the molecular mechanism of proton translocation, it is therefore essential to establish the structure and function of domain II and its interactions with domains I and III. This review describes some relevant recent results in this field of research
Functional split and crosslinking of the membrane domain of the beta subunit of proton-translocating transhydrogenase from Escherichia coli
No full textProton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an alpha subunit with the NAD(H)-binding domain I and a beta subunit with the NADP(H)-binding domain III. The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic parts of the alpha and beta subunits. The interface in domain II between the alpha and the beta subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the alpha subunit and loops connecting the nine transmembrane helices in the beta subunit. However, to investigate the organization of the nine transmembrane helices in the beta subunit, a split was introduced by creating a stop codon in the loop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existing downstream start codon. The resulting enzyme was composed of the wild-type alpha subunit and the two new peptides beta1 and beta2. As compared to other split membrane proteins, the new transhydrogenase was remarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD(+) by NADPH, the cyclic reduction of 3-acetylpyridine-NAD(+) by NADH (mediated by bound NADP(H)), and proton pumping, amounting to about 50-107% of the corresponding wild-type activities. These high activities suggest that the alpha subunit was normally folded, followed by a concerted folding of beta1 + beta2. Cross-linking of a betaS105C-betaS237C double cysteine mutant in the functional split cysteine-free background, followed by SDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity. This is the first time that cross-linking between helices in the same beta subunit has been demonstrated
