| 1. |
- Lundbäck, Anna-Karin, et al.
(författare)
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Assembly of Kch, a putative potassium channel from Escherichia coli
- 2009
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Ingår i: Journal of Structural Biology. - : Elsevier BV. - 1047-8477 .- 1095-8657. ; 168:2, s. 288-293
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Tidskriftsartikel (refereegranskat)abstract
- Attempts to explore the structure and function of Kch, a putative potassium channel of Escherichia coli have yielded varying results; potassium-associated functions have been found in vivo but not in vitro. Here the kch gene is shown to produce two proteins, full-length Kch and the large C-terminal cytosolic domain (the RCK domain). Further, these two proteins are associated at the initial stages of purification. Previous structural studies of full-length Kch claim that the isolated protein forms large aggregates that are not suitable for analysis. The results presented here show that the purified protein sample, although heterogeneous, has one major population with a mass of about 400 kDa, implying the presence of two Kch tetramers in a complex form. A three dimensional reconstruction at 25 angstrom based on electron microscopy data from negatively stained particles, revealed a 210 angstrom long and 95 angstrom wide complex in which the two tetrameric Kch units are linked by their RCK domains, giving rise to a large central ring of density. The formation of this dimer of tetramers on expression or during purification, may explain why attempts to reconstitute Kch into liposomes for activity measurements have failed.
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| 2. |
- Lundbäck, Anna-Karin, et al.
(författare)
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Exploring the activity of tobacco etch virus protease in detergent solutions
- 2008
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 382:1, s. 69-71
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Tidskriftsartikel (refereegranskat)abstract
- Tobacco etch virus (TEV) protease is generally used to remove affinity tags from target proteins. It has been reported that some detergents inhibit the activity of this protease, and therefore should be avoided when removing affinity tags from membrane proteins. The aim of this study was to explore and evaluate this further. Hence, affinity tag removal with TEV protease was tested from three membrane proteins (a Pgp synthase and two CorA homologs) in the presence of 16 different detergents commonly used in membrane protein purification and crystallization. We observed that in the presence of the same detergent (Triton X-100), TEV protease could remove the affinity tag completely from one protein (CorA) but not from another protein (Pgp synthase). There was also a large variation in yield of cleaved membrane protein in different detergents, which probably depends on features of the protein-detergent complex. These observations show that, contrary to an earlier report, detergents do not inhibit the enzymatic activity of the TEV protease.
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| 3. |
- Lundbäck, Anna-Karin
(författare)
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Understanding structure and function of membrane protein transporters
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membrane protein transporters are important proteins in the cell, as they maintain different solutes and metabolites at a stable concentration. Numerous diseases can be linked to malfunctioning transporters, which makes them interesting pharmaceutical targets. To learn more how these proteins are regulated both functional and structural studies are needed. Membrane proteins are challenging since it is not easy to produce large amounts of these proteins and they readily precipitate due to their hydrophobic nature. In this thesis the function or structure of three different membrane protein transporters were studied; Melibiose permease (MelB), a sugar co-transporter from Escherichia coli, a potassium channel (Kch) from Escherichia coli and a divalent ion transporter from Thermotoga maritima (CorA). Studies of two-dimensional crystals from MelB with Cryo electron microscopy resulted in a three-dimensional structure to 10 Å. The structure revealed an asymmetrical cone shaped molecule that clearly was closed in one end and open in the other. The overall structure resembled that of the Na+/H+ antiporter (NhaA) more than that of the lactose permease (LacY). Preliminary results from crystals without substrate show structural differences in the projection map. Also crystals without substrate were obtained, which preliminary show structural differences in the projection map. Single particle studies of Kch indicated that the majority of tetramers of the protein probably dimerize in a way that can explain why the protein has been difficult to reconstitute into liposomes. Functional studies of CorA from Thermotoga maritima demonstrated that the substrate may be Co2+, and thus Mg2+ may not be the primary substrate of all CorA proteins as previously anticipated. Furthermore fluorescence measurements of tryptophan quenching indicated that there are two binding sites for Co2+ with different affinities (Kd values was 28 μM and 750 μM). During these studies methods to obtain stable protein were investigated. A small-scale approach for a buffer screen with analytical size exclusion chromatography was developed. With this method a protein, that previously precipitated during ultrafiltration was possible to concentrate to amounts suitable for crystallization trials. Also, the activity of TEV protease was investigated in different detergents. This protease is commonly used to remove affinity tags from proteins. Our results show that the choice of the detergent affects the accessibility of the membrane protein cleavage site and not the activity of the TEV protease. Taken together; the results presented in this thesis deals with problems of stability of purified membrane proteins and protease activity it detergent solutions. Structures of two membrane proteins (MelB and Kch) were determined with two different electron microscopy methods, and the substrate specificity of CorA was explored and revised
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| 4. |
- Lundbäck, Peter, et al.
(författare)
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A novel high mobility group box 1 neutralizing chimeric antibody attenuates drug-induced liver injury and postinjury inflammation in mice
- 2016
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Ingår i: Hepatology. - : Ovid Technologies (Wolters Kluwer Health). - 0270-9139 .- 1527-3350. ; 64:5, s. 1699-1710
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Tidskriftsartikel (refereegranskat)abstract
- Acetaminophen (APAP) overdoses are of major clinical concern. Growing evidence underlines a pathogenic contribution of sterile postinjury inflammation in APAP-induced acute liver injury (APAP-ALI) and justifies development of anti-inflammatory therapies with therapeutic efficacy beyond the therapeutic window of the only current treatment option, N-acetylcysteine (NAC). The inflammatory mediator, high mobility group box 1 (HMGB1), is a key regulator of a range of liver injury conditions and is elevated in clinical and preclinical APAP-ALI. The anti-HMGB1 antibody (m2G7) is therapeutically beneficial in multiple inflammatory conditions, and anti-HMGB1 polyclonal antibody treatment improves survival in a model of APAP-ALI. Herein, we developed and investigated the therapeutic efficacy of a partly humanized anti-HMGB1 monoclonal antibody (mAb; h2G7) and identified its mechanism of action in preclinical APAP-ALI. The mouse anti-HMGB1 mAb (m2G7) was partly humanized (h2G7) by merging variable domains of m2G7 with human antibody-Fc backbones. Effector function-deficient variants of h2G7 were assessed in comparison with h2G7 in vitro and in preclinical APAP-ALI. h2G7 retained identical antigen specificity and comparable affinity as m2G7. 2G7 treatments significantly attenuated APAP-induced serum elevations of alanine aminotransferase and microRNA-122 and completely abrogated markers of APAP-induced inflammation (tumor necrosis factor, monocyte chemoattractant protein 1, and chemokine [C-X-C motif] ligand 1) with prolonged therapeutic efficacy as compared to NAC. Removal of complement and/or Fc receptor binding did not affect h2G7 efficacy. Conclusion: This is the first report describing the generation of a partly humanized HMGB1-neutralizing antibody with validated therapeutic efficacy and with a prolonged therapeutic window, as compared to NAC, in APAP-ALI. The therapeutic effect was mediated by HMGB1 neutralization and attenuation of postinjury inflammation. These results represent important progress toward clinical implementation of HMGB1-specific therapy as a means to treat APAP-ALI and other inflammatory conditions. (Hepatology 2016;64:1699-1710).
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| 5. |
- Molina, Daniel Martinez, et al.
(författare)
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Expression and purification of the recombinant membrane protein YidC : A case studyfor increased solubility and stability
- 2008
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 62:1, s. 49-52
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Tidskriftsartikel (refereegranskat)abstract
- YidC is an inner membrane protein from Escherichia coli and is an essential component in insertion, trans- location and assembly of membrane proteins in the membranes. Previous purification attempts resulted in heavy aggregates and precipitated protein at later stages of purification. Here we present a rapid and straightforward stability screening strategy based on gel filtration chromatography, which requires as little as 10 lg of protein and takes less than 15 min to perform. With this technique, we could rapidly screen several buffers in order to identify an optimum condition that stabilizes purified YidC. After optimization we could obtain several milligrams of purified YidC that could be easily prepared at high con- centrations and that was stable for weeks at +4 C. The isolated protein is thus well suited for structural studies.
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| 6. |
- Xia, Yu, et al.
(författare)
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Co(2+) Selectivity of Thermotoga maritima CorA and Its Inability to Regulate Mg(2+) Homeostasis Present a New Class of CorA Proteins
- 2011
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 286:18
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Tidskriftsartikel (refereegranskat)abstract
- CorA is a family of divalent cation transporters ubiquitously present in bacteria and archaea. Although CorA can transport both Mg(2+) and Co(2+) almost equally well, its main role has been suggested to be that of primary Mg(2+) transporter of prokaryotes and hence the regulator of Mg(2+) homeostasis. The reason is that the affinity of CorA for Co(2+) is relatively low and thus considered non-physiological. Here, we show that Thermotoga maritima CorA (TmCorA) is incapable of regulating the Mg(2+) homeostasis and therefore cannot be the primary Mg(2+) transporter of T. maritima. Further, our in vivo experiments confirm that TmCorA is a highly selective Co(2+) transporter, as it selects Co(2+) over Mg(2+) at > 100 times lower concentrations. In addition, we present data that show TmCorA to be extremely thermostable in the presence of Co(2+). Mg(2+) could not stabilize the protein to the same extent, even at high concentrations. We also show that addition of Co(2+), but not Mg(2+), specifically induces structural changes to the protein. Altogether, these data show that TmCorA has the role of being the transporter of Co(2+) but not Mg(2+). The physiological relevance and requirements of Co(2+) in T. maritima is discussed and highlighted. We suggest that CorA may have different roles in different organisms. Such functional diversity is presumably a reflection of minor, but important structural differences within the CorA family that regulate the gating, substrate selection, and transport.
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