| 1. |
|
|
| 2. |
- Althage, Magnus, et al.
(författare)
-
Cross-linking of transmembrane helices in proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli: implications for the structure and function of the membrane domain.
- 2004
-
Ingår i: Biochimica et biophysica acta. - : Elsevier BV. - 0006-3002 .- 0005-2728. ; 1659:1, s. 73-82
-
Tidskriftsartikel (refereegranskat)abstract
- Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an alpha and a beta 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 alpha-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 A), and visualized by SDS-PAGE. In the alpha(2)beta(2) tetramer, alphabeta cross-links were obtained with the alphaG476C-betaS2C, alphaG476C-betaT54C and alphaG476C-betaS183C double mutants. Significant alphaalpha cross-links were obtained with the alphaG476C single mutant in the loop connecting helix 3 and 4, whereas betabeta cross-links were obtained with the betaS2C, betaT54C and betaS183C 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 alpha and beta subunits in the dimer is lined along an axis formed by helices 3 and 4 from the alpha subunit and helices 6, 7 and 8 from the beta subunit. In addition, helices 2 and 4 in the alpha subunit together with helices 6 and 12 in the beta subunit interact with their counterparts in the alpha(2)beta(2) tetramer. Each beta subunit in the alpha(2)beta(2) tetramer was concluded to contain a proton channel composed of the highly conserved helices 9, 10, 13 and 14.
|
|
| 3. |
- Arkblad, Eva L, et al.
(författare)
-
A Caenorhabditis elegans mutant lacking functional nicotinamide nucleotide transhydrogenase displays increased sensitivity to oxidative stress.
- 2005
-
Ingår i: Free radical biology & medicine. - : Elsevier BV. - 0891-5849. ; 38:11, s. 1518-25
-
Tidskriftsartikel (refereegranskat)abstract
- Proton-translocating mitochondrial nicotinamide nucleotide transhydrogenase (NNT) was investigated regarding its physiological role in Caenorhabditis elegans. NNT catalyzes the reduction of NADP(+) by NADH driven by the electrochemical proton gradient, Deltap, and is thus a potentially important source of mitochondrial NADPH. Mitochondrial detoxification of reactive oxygen species (ROS) by glutathione-dependent peroxidases depends on NADPH for regeneration of reduced glutathione. Transhydrogenase may therefore be directly involved in the defense against oxidative stress. nnt-1 deletion mutants of C. elegans, nnt-1(sv34), were isolated and shown to grow essentially as wild type under normal laboratory conditions, but with a strongly lowered GSH/GSSG ratio. Under conditions of oxidative stress, caused by the superoxide-generating agent methyl viologen, growth of worms lacking nnt-1 activity was severely impaired. A similar result was obtained by using RNAi. Reintroducing nnt-1 in the nnt-1(sv34) knockout mutant led to a partial rescue of growth under oxidative stress conditions. These results provide evidence for the first time that nnt-1 is important in the defense against mitochondrial oxidative stress.
|
|
| 4. |
- Egorov, Maxim V, et al.
(författare)
-
Purification of a recombinant membrane protein tagged with a calmodulin-binding domain: properties of chimeras of the Escherichia coli nicotinamide nucleotide transhydrogenase and the C-terminus of human plasma membrane Ca2+ -ATPase.
- 2004
-
Ingår i: Protein expression and purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 36:1, s. 31-9
-
Tidskriftsartikel (refereegranskat)abstract
- A Ca2+ -dependent calmodulin-binding peptide (CBP) is an attractive tag for affinity purification of recombinant proteins, especially membrane proteins, since elution is simply accomplished by removing/chelating Ca2+. To develop a single-step calmodulin/CBP-dependent purification procedure for Escherichia coli nicotinamide nucleotide transhydrogenase, a 49 amino acid large CBP or a larger 149 amino acid C-terminal fragment of human plasma membrane Ca2+ -ATPase (hPMCA) was fused C-terminally to the beta subunit of transhydrogenase. Fusion using the 49 amino acid fragment resulted in a dramatic loss of transhydrogenase expression while fusion with the 149 amino acid fragment gave a satisfactory expression. This chimeric protein was purified by affinity chromatography on calmodulin-Sepharose with mild elution with EDTA. The purity and activity were comparable to those obtained with His-tagged transhydrogenase and showed an increased stability. CBP-tagged transhydrogenase contained a 4- to 10-fold higher amount of the alpha subunit relative to the beta subunit as compared to wild-type transhydrogenase. To determine whether the latter was due to the CBP tag, a double-tagged transhydrogenase with both an N-terminal 6x His-tag and a CBP-tag, purified by using either tag, gave no significant increase in purity as compared to the single-tagged protein. The reasons for the altered subunit composition are discussed. The results suggest that, depending on the construct, the CBP-tag may be a suitable affinity purification tag for membrane proteins in general.
|
|
| 5. |
|
|
| 6. |
|
|
| 7. |
|
|
| 8. |
- Graneli, Annette, 1973, et al.
(författare)
-
Formation of Supported Lipid Bilayer Membranes on SiO2 from Proteoliposomes Containing Transmembrane Proteins
- 2003
-
Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 19, s. 842-850
-
Tidskriftsartikel (refereegranskat)abstract
- We report the preparation of protein-containing supported phospholipid bilayers (SPBs) on silica (SiO2). The bilayers are formed from small proteoliposomes, which convert to an SPB when the liposomes adsorb on the surface. The kinetics of this conversion process was followed in real time, using the quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) techniques. The proteoliposomes were prepared by reconstitution of two different proteins into small unilamellar liposomes (diameter ~ 26 nm), creating proteoliposomes with diameters ranging from ca. 50 to 85 nm, depending on protein concentration. The two proteins were proton translocating nicotinamide nucleotide transhydrogenase (TH) from Escherichia coli and gramicidin A (GrA) from Bacillus brevis. The SPB formation process was measured and compared for different protein situations in the liposomes: (i) with the intact TH in the proteoliposomes, (ii) after removal of the water-exposed, hydrophilic domains of TH, and (iii) with GrA-containing proteoliposomes (with no water-soluble domains). In the latter two cases qualitatively similar kinetics were observed as with pure (i.e., without proteins) liposomes. In contrast, the water-exposed hydrophilic domains on TH are found to partially hamper the SPB formation process leaving fractions of nonruptured proteoliposomes on the surface. The latter effect becomes stronger with increasing protein/lipid ratio in the proteoliposomes. A comparison was made between activity measurements of TH-containing proteoliposomes in solution and TH-containing SPBs. The latter results support the conclusions from the QCM-D and SPR measurements.
|
|
| 9. |
- Granéli, Annette, 1973, et al.
(författare)
-
Utilizing adsorbed proteoliposomes trapped in a non-ruptured state on SiO2 for amplified detection of membrane proteins
- 2004
-
Ingår i: Biosensors & bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 20:3, s. 498-504
-
Tidskriftsartikel (refereegranskat)abstract
- The quartz crystal microbalance with dissipation (QCM-D) technique was used to monitor the formation of supported phospholipid bilayers (SPBs) on SiO2 using proteoliposomes with reconstituted proton translocating nicotinamide nucleotide transhydrogenase (TH). Exposure of the surface to such proteoliposomes creates a lipid film composed of a mixture of proteolipid bilayers and adsorbed non-ruptured proteoliposomes, where the fraction of the latter is reduced if the TH-liposomes are pretreated with trypsin to remove the water soluble domains of TH [Langmuir 19 (2003) 842]. In the present work, the latter study is complemented by investigating the influence of trypsin treatment of the mixed adlayer (proteolipid bilayer + non-ruptured proteoliposomes) after adsorption on the surface. This demonstrates how trypsin-cleavage induced rupture of adsorbed TH-liposomes can be utilized to detect the presence of less than 0.04 pmol/cm2 of immobilized TH.
|
|
| 10. |
- Hedfalk, Kristina, 1969, et al.
(författare)
-
A Regulatory Domain in the C-terminal Extension of the Yeast Glycerol Channel Fps1p
- 2004
-
Ingår i: Journal of biological chemistry. ; 279:15, s. 14954-14960
-
Tidskriftsartikel (refereegranskat)abstract
- The Saccharomyces cerevisiae gene FPS1 encodes an aquaglyceroporin of the major intrinsic protein (MIP) family. The main function of Fps1p seems to be the efflux of glycerol in the adaptation of the yeast cell to lower external osmolarity. Fps1p is an atypical member of the family, because the protein is much larger (669 amino acids) than most MIPs due to long hydrophilic extensions in both termini. We have shown previously that a short domain in the N-terminal extension of the protein is required for restricting glycerol transport through the channel (Tamás, M. J., Karlgren, S., Bill, R. M., Hedfalk, K., Allegri, L., Ferreira, M., Thevelein, J. M., Rydström, J., Mullins, J. G. L., and Hohmann, S. (2003) J. Biol. Chem. 278, 63376345). Deletion of the N-terminal domain results in an unregulated channel, loss of glycerol, and osmosensitivity. In this work we have investigated the role of the Fps1p C terminus (139 amino acids). A set of eight truncations has been constructed and tested in vivo in a yeast fps1 strain. We have performed growth tests, membrane localization following cell fractionation, and glycerol accumulation measurements as well as an investigation of the osmotic stress response. Our results show that the C-terminal extension is also involved in restricting transport through Fps1p. We have identified a sequence of 12 amino acids, residues 535546, close to the sixth transmembrane domain. This element seems to be important for controlling Fps1p function. Similar to the N-terminal domain, the C-terminal domain is amphiphilic and has a potential to dip into the membrane
|
|
