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1.
Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential ('proton-motive force'), either by transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by transmembrane proton transfer. Here we provide the first evidence that both of these mechanisms are combined in the case of a specific respiratory membrane protein complex, the dihaem-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes, so as to facilitate transmembrane electron transfer by transmembrane proton transfer. We also demonstrate the non-functionality of this novel transmembrane proton transfer pathway ('E-pathway') in a variant QFR where a key glutamate residue has been replaced. The 'E-pathway', discussed on the basis of the 1.78-Angstrom-resolution crystal structure of QFR, can be concluded to be essential also for the viability of pathogenic varepsilon-proteobacteria such as Helicobacter pylori and is possibly relevant to proton transfer in other dihaem-containing membrane proteins, performing very different physiological functions. 相似文献
2.
Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential (Δp), either by supporting transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by supporting transmembrane proton transfer. The first mechanism has been unequivocally demonstrated to be operational for Δp-dependent catalysis of succinate oxidation by quinone in the case of the dihaem-containing succinate:menaquinone reductase (SQR) from the Gram-positive bacterium Bacillus licheniformis. This is physiologically relevant in that it allows the transmembrane potential Δp to drive the endergonic oxidation of succinate by menaquinone by the dihaem-containing SQR of Gram-positive bacteria. In the case of a related but different respiratory membrane protein complex, the dihaem-containing quinol:fumarate reductase (QFR) of the ?-proteobacterium Wolinella succinogenes, evidence has been obtained that both mechanisms are combined, so as to facilitate transmembrane electron transfer by proton transfer via a both novel and essential compensatory transmembrane proton transfer pathway (“E-pathway”). Although the reduction of fumarate by menaquinol is exergonic, it is obviously not exergonic enough to support the generation of a Δp. This compensatory “E-pathway” appears to be required by all dihaem-containing QFR enzymes and results in the overall reaction being electroneutral. However, here we show that the reverse reaction, the oxidation of succinate by quinone, as catalysed by W. succinogenes QFR, is not electroneutral. The implications for transmembrane proton transfer via the E-pathway are discussed. 相似文献
3.
Cytochromes b(561) are a family of transmembrane proteins found in most eukaryotic cells and contain two haem b prosthetic groups per molecule being coordinated with four His residues from four different transmembrane alpha-helices. Although cytochromes b(561) residing in the chromaffin vesicles has long been known to have a role for a neuroendocrine-specific transmembrane electron transfer from extravesicular ascorbate to intravesicular monodehydroascorbate radical to regenerate ascorbate, newly found members were apparently lacking in the sequence for putative ascorbate-binding site but exhibiting a transmembrane ferrireductase activity. We propose that cytochrome b(561) has a specific mechanism to facilitate the concerted proton/electron transfer from ascorbate by exploiting a cycle of deprotonated and protonated states of the N(delta1) atom of the axial His residue at the extravesicular haem center, as an initial step of the transmembrane electron transfer. This mechanism utilizes the well-known electrochemistry of ascorbate for a biological transmembrane electron transfer and might be operative for other type of electron transfer reactions from organic reductants. 相似文献
4.
Polytopic protein topology is established in the endoplasmic reticulum (ER) by sequence determinants encoded throughout the nascent polypeptide. Here we characterize 12 topogenic determinants in the cystic fibrosis transmembrane conductance regulator, and identify a novel mechanism by which a charged residue is positioned within the plane of the lipid bilayer. During cystic fibrosis transmembrane conductance regulator biogenesis, topology of the C-terminal transmembrane domain (TMs 7-12) is directed by alternating signal (TMs 7, 9, and 11) and stop transfer (TMs 8, 10, and 12) sequences. Unlike conventional stop transfer sequences, however, TM8 is unable to independently terminate translocation due to the presence of a single charged residue, Asp(924), within the TM segment. Instead, TM8 stop transfer activity is specifically dependent on TM7, which functions both to initiate translocation and to compensate for the charged residue within TM8. Moreover, even in the presence of TM7, the N terminus of TM8 extends significantly into the ER lumen, suggesting a high degree of flexibility in establishing TM8 transmembrane boundaries. These studies demonstrate that signal sequences can markedly influence stop transfer behavior and indicate that ER translocation machinery simultaneously integrates information from multiple topogenic determinants as they are presented in rapid succession during polytopic protein biogenesis. 相似文献
5.
V V Khramtsov M V Panteleev L M Weiner 《Journal of biochemical and biophysical methods》1989,18(3):237-246
A new method for measuring the rates of proton transfer through bilayer phospholipid membranes using pH-sensitive nitroxyl radicals is suggested. The pH-sensitive alkylating radical was covalently bound to glutathione. This modified glutathione is pH sensitive at pH 1.5-4.5 and does not penetrate across phospholipid membranes. Using ESR this probe was applied to register the kinetics of pH variations inside large unilamellar phospholipid vesicles after creation of a transmembrane proton gradient. In the acidic region (pH approximately 3) the main mechanism of transmembrane proton transfer is that via transport of a proton in the form of an undissociated acid. The membrane permeability coefficients have been determined for a series of acids (HCl, HClO4, HNO3, upper estimate for H2SO4). Taking into account that imidazoline and imidazolidine nitroxyl radicals can be used as pH probes in a wide range of pH, the present method can be developed for measuring the rates of transmembrane proton transfer in neutral and alkaline media. 相似文献
6.
Fluorescence resonance energy transfer measurements reveal that a transmembrane sequence within a nascent membrane protein folds into a compact conformation near the peptidyltransferase center and remains folded as the sequence moves through a membrane bound ribosome into the translocon. This compact conformation is compatible with an alpha helix because nearly the same energy transfer efficiency was observed when the transmembrane sequence was integrated into the lipid bilayer. Since the transmembrane sequence unfolds upon emerging from a free ribosome, this nascent chain folding is ribosome induced and stabilized. In contrast, a nascent secretory protein is in an extended conformation in the exit tunnel. Furthermore, two ribosomal proteins photo-crosslink to nascent membrane but not secretory proteins. These interactions coincide with the previously described sequential closing and opening of the two ends of the aqueous translocon pore, thereby suggesting that ribosomal recognition of nascent chain folding controls the operational mode of the translocon at the ER membrane. 相似文献
7.
Cytochrome b561 from bovine adrenal medulla chromaffin granules has been purified by fast protein liquid chromatography chromatofocusing. The purified cytochrome was reconstituted into ascorbate-loaded phosphatidylcholine vesicles. With this reconstituted system transmembrane electron transfer for extravesicular soluble dopamine beta-hydroxylase activity was demonstrated. In accordance with the model proposed by Njus et al. (Njus, D., Knoth, J., Cook, C., and Kelley, P. M. (1983) J. Biol. Chem. 258, 27-30), catalytic amounts of a redox mediator were necessary to achieve electron transfer between cytochrome and soluble dopamine beta-hydroxylase. Our observations also showed that when membranous dopamine beta-hydroxylase was reconstituted on cytochrome containing vesicles, electron transfer occurred only in the presence of a redox mediator. Since cytochrome b561 has been found in secretory vesicles associated with peptidyl glycine alpha-amidating monooxygenase, electron transfer to this enzyme was also examined. Analogous to the results obtained for dopamine beta-hydroxylase, transmembrane electron transfer to peptidyl glycine alpha-amidating monooxygenase appears to require a redox mediator between cytochrome and this monooxygenase. These observations indicate that purified cytochrome b561 is capable of providing a transmembrane supply of electrons for both monooxygenases. Since no direct protein to protein electron transfer occurs, the results support the hypothesis that the ascorbate/semidehydroascorbate redox pair serves as a mediator for these enzymes in vivo. 相似文献
8.
Ujihara S Oishi T Torikai K Konoki K Matsumori N Murata M Oshima Y Aimoto S 《Bioorganic & medicinal chemistry letters》2008,18(23):6115-6118
Ladder-shaped polyether (LSP) compounds are thought to interact with transmembrane alpha-helices, but direct evidence has scarcely obtained for these interactions. We adopted a transmembrane alpha-helix of glycophorin A, and quantitatively evaluated its interaction with LSPs such as yessotoxin (YTX), desulfated YTX and artificial LSPs, using surface plasmon resonance and saturation transfer difference NMR. As a result, dissociation constants (K(D)) of YTX and desulfated YTX to a transmembrane domain peptide of glycophorin A were determined to be in the submillimolar range. Furthermore, in saturation transfer difference NMR, the signals at the polyene side chain and the angular methyl groups of YTX were significantly attenuated, which probably comprised an interacting interface of LSPs with a transmembrane alpha-helix. These results suggest that hydrophobic interaction plays an important role in molecular recognition of the alpha-helix peptide by LSPs. 相似文献
9.
Lancaster CR 《Biochimica et biophysica acta》2002,1565(2):215-231
The structure of the respiratory membrane protein complex quinol:fumarate reductase (QFR) from Wolinella succinogenes has been determined by X-ray crystallography at 2.2-A resolution [Nature 402 (1999) 377]. Based on the structure of the three protein subunits A, B, and C and the arrangement of the six prosthetic groups (a covalently bound FAD, three iron-sulfur clusters, and two haem b groups), a pathway of electron transfer from the quinol-oxidising dihaem cytochrome b in the membrane to the site of fumarate reduction in the hydrophilic subunit A has been proposed. The structure of the membrane-integral dihaem cytochrome b reveals that all transmembrane helical segments are tilted with respect to the membrane normal. The "four-helix" dihaem binding motif is very different from other dihaem-binding transmembrane four-helix bundles, such as the "two-helix motif" of the cytochrome bc(1) complex and the "three-helix motif" of the formate dehydrogenase/hydrogenase group. The gamma-hydroxyl group of Ser C141 has an important role in stabilising a kink in transmembrane helix IV. By combining the results from site-directed mutagenesis, functional and electrochemical characterisation, and X-ray crystallography, a residue was identified which was found to be essential for menaquinol oxidation [Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 13051]. The distal location of this residue in the structure indicates that the coupling of the oxidation of menaquinol to the reduction of fumarate in dihaem-containing succinate:quinone oxidoreductases could in principle be associated with the generation of a transmembrane electrochemical potential. However, it is suggested here that in W. succinogenes QFR, this electrogenic effect is counterbalanced by the transfer of two protons via a proton transfer pathway (the "E-pathway") in concert with the transfer of two electrons via the membrane-bound haem groups. According to this "E-pathway hypothesis", the net reaction catalysed by W. succinogenes QFR does not contribute directly to the generation of a transmembrane electrochemical potential. 相似文献
10.
A I Vinnikov 《Ukrainski? biokhimicheski? zhurnal》1987,59(6):50-54
The generation of transmembrane difference of electrochemical potentials was registered on the intact cells and ultrasonication-obtained membrane vesicles of Staphylococcus aureus with the application of transmembrane electrophoresis of permeant anions, potassium transport in the presence of valinomycin and 8-anilinonaphthalene-1-sulphonate fluorescence. The membrane potential is formed when the chain of electron transfer or H+-ATPase functions or when the pH gradient varies (the nonenzymic pathway). M-chlorinecarbonylcyanidephenylhydrazonium, a protonophore uncoupler potassium cyanide, an inhibitor of the respiratory chain, N',N-dicyclohexylcarbodiimide, an inhibitor of ATPase, cause the membrane potential dissipation. The orientation of the transmembrane electric field is as follows: "minus" inside cells and "plus" inside membrane vesicles. 相似文献
11.
De novo protein design has created small, robust protein-cofactor complexes that serve as simplified working models, or maquettes, for the much more complicated natural oxidoreductases. We review the research avenues that spring from the better characterized water-soluble hydrophilic maquettes and guide the construction of amphiphilic maquettes patterned on membrane-bound oxidoreductases that couple electron transfer to transmembrane proton-motive force. We address the special working challenges and opportunities that arise with amphiphilic maquettes designed to assemble in membranes, along with the redox and pigment cofactors required to promote light activated electron transfer, redox-coupled electric field generation, proton exchange and transmembrane charge motion. 相似文献
12.
An important subject for elucidating membrane protein (MP) folding is how transmembrane helices (TMHs) insert into and dissociate from membranes. We investigated helix dissociation kinetics and insertion topology by means of intervesicular transfer of the fluorophore-labeled completely hydrophobic model transmembrane helix NBD-(LALAAAA)(3)-NH(2) (NBD = 7-nitro-2-1,3-benzoxadiazol-4-yl). The peptide forms a topologically stable transmembrane helix, which is in a monomer-antiparallel dimer equilibrium [Yano, Y., Takemoto, T., Kobayashi, S., Yasui, H., Sakurai, H., Ohashi, W., Niwa, M., Futaki, S., Sugiura, Y., and Matsuzaki, K. (2002) Biochemistry 41, 3073-3080]. The helix transfer kinetics, representing the helix dissociation process, was monitored by fluorescence recovery of the quenched peptide in donor vesicles containing a quencher upon its transfer to acceptor vesicles without the quencher. The transfer kinetics and vesicle concentration dependence demonstrated that the transfer was mediated by monomer in the aqueous phase. Furthermore, the activation enthalpy was estimated to be +17.7 +/- 1.3 kcal mol(-1). Helix insertion topology, detected by chemical quenching of the NBD group in the outer leaflet by dithionite ions, was found to be controlled by transmembrane electric potential-helix macro dipole interaction. On the basis of these observations, a model for the helix insertion/dissociation processes was discussed. 相似文献
13.
14.
Trans-membrane translocation of proteins. The direct transfer model. 总被引:33,自引:0,他引:33
As a start towards a deeper understanding of the transmembrane transport of proteins, the transfer of a nascent chain through the lipophilic core of a membrane is discussed from a physico-chemical point of view. Some simple considerations of the energetics of protein structure, together with experimental data on the transfer process, form the basis for a detailed and quantifiable model, accounting for the extrusion of secreted proteins as well as for the insertion of trans-membrane proteins. 相似文献
15.
Direct effects of ionizing radiation on integral membrane proteins. Noncovalent energy transfer requires specific interpeptide interactions 总被引:1,自引:0,他引:1
The 12 transmembrane alpha helices (TMHs) of human erythrocyte glucose transporter were individually cut by pepsin digestion as membrane-bound 2.5-3.5-kDa peptide fragments. Radiation-induced chemical degradation of these fragments showed an average target size of 34 kDa. This is 10-12 x larger than the average size of an individual TMH, demonstrating that a significant energy transfer occurs among these TMHs in the absence of covalent linkage. Heating this TMH preparation at 100 degrees C for 15 min reduced the target size to 5 kDa or less, suggesting that the noncovalent energy transfer requires specific helix-helix interactions. Purified phospholamban, a small (6-kDa) integral membrane protein containing a single TMH, formed a pentameric assembly in sodium dodecyl sulfate. The chemical degradation target size of this phospholamban pentamer was 5-6 kDa, illustrating that not all integral membrane protein assemblies permit intersubunit energy transfer. These findings together with other published observations suggest strongly that significant noncovalent energy transfer can occur within the tertiary and quaternary structure of membrane proteins and that as yet undefined proper molecular interactions are required for such covalent energy transfer. Our results with pepsin-digested glucose transporter also illustrate the importance of the interhelical interaction as a predominating force in maintaining the tertiary structure of a transmembrane protein. 相似文献
16.
The influence of ferricyanide on transmembrane electron transfer,proton secretion, membrane potential, and cytoplasmic pH ofLimnobium stoloniferum (G.F. Mey) Griseb. root cells was investigatedat different external pH HCF III reduction by the roots was accompanied by membrane depolarization,an increase in proton secretion and by alkalinization of thecytoplasm. Change of membrane potential and cytoplasmic pH aswell as transmembrane e transfer was more pronouncedat acid external pH. The rate of proton flux was linearly dependenton the rate of electron transfer. The slope of the relationshipwas around 1, independent of external pH The data are in agreement with the hypothesis that electrontransfer at the plasma membrane is directly coupled to protonsecretion. It is suggested that both e and redox-coupledH+ transport are activated by acid external pH Key words: Plasmalemma redox system, electron transfer, proton transport, pH, membrane potential, Limnobium stoloniferum 相似文献
17.
Model for growth hormone receptor activation based on subunit rotation within a receptor dimer 总被引:8,自引:0,他引:8
Brown RJ Adams JJ Pelekanos RA Wan Y McKinstry WJ Palethorpe K Seeber RM Monks TA Eidne KA Parker MW Waters MJ 《Nature structural & molecular biology》2005,12(9):814-821
Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand. 相似文献
18.
Sansom MS 《Current biology : CB》2000,10(5):R206-R209
The fourth transmembrane helix (S4) is the primary voltage-sensor of voltage-gated ion channels. Recent studies have used fluorescence resonance energy transfer as a spectroscopic ruler to determine the nature and magnitude of the voltage-induced movement of S4 that leads to channel opening. 相似文献
19.
I A Gorskaia O D Kochergina Iu N Antonenko A V Kotel'nikova L S Iaguzhinski? 《Biokhimii?a (Moscow, Russia)》1984,49(5):821-826
The transmembrane reaction of ferricyanide reduction by exogenous ascorbate in the liposomes in the presence of N,N,N',N'-tetramethylparaphenylenediamine (TMPD) or 2,3,5,6-tetramethylphenylenediamine (DAD) was investigated. The reaction equilibrium was shown to depend on the intraliposomal pH. At alkaline pH values under the experimental conditions used TMPD functions mainly as an electron carrier, while at acidic pH values TMPD effectuates a coupled transmembrane electron and proton transfer. This reaction is paralleled with local changes in the pH values in the unstirred layer near the membrane. 相似文献
20.
The cytochrome c oxidase complex (CcO) catalyzes the four-electron reduction of dioxygen to water by using electrons from ferrocytochrome c. Redox free energy released in this highly exergonic process is utilized to drive the translocation of protons across a transmembrane electrochemical gradient. Although numerous chemical models of proton pumping have been developed, few attempts have been made to explain the stepwise transfer of energy in the context of proposed protein conformational changes. A model is described that seeks to clarify the thermodynamics of the proton pumping function of CcO and that illustrates the importance of electron and proton gating to prevent the occurrence of the more exergonic electron leak and proton slip reactions. The redox energy of the CcO-membrane system is formulated in terms of a multidimensional energy surface projected into two dimensions, a nuclear coordinate associated with electron transfer and a nuclear coordinate associated with elements of the proton pump. This model provides an understanding of how a transmembrane electrochemical gradient affects the efficiency of the proton pumping process. Specifically, electron leak and proton slip reactions become kinetically viable as a result of the greater energy barriers that develop for the desired reactions in the presence of a transmembrane potential. 相似文献