| 1. |
- Backmark, Anna, 1979, et al.
(författare)
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Affinity tags can reduce merohedral twinning of membrane protein crystals
- 2008
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; D64, s. 1183-1186
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Tidskriftsartikel (refereegranskat)abstract
- This work presents a comparison of the crystal packing of three eukaryotic membrane proteins: human aquaporin 1, human aquaporin 5 and a spinach plasma membrane aquaporin. All were purified from expression constructs both with and without affinity tags. With the exception of tagged aquaporin 1, all constructs yielded crystals. Two significant effects of the affinity tags were observed: crystals containing a tag typically diffracted to lower resolution than those from constructs encoding the protein sequence alone and constructs without a tag frequently produced crystals that suffered from merohedral twinning. Twinning is a challenging crystallographic problem that can seriously hinder solution of the structure. Thus, for integral membrane proteins, the addition of an affinity tag may help to disrupt the approximate symmetry of the protein and thereby reduce or avoid merohedral twinning.
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| 2. |
- Elbing, Karin, 1974, et al.
(författare)
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Towards the structure-function-specificity relationship of glucose transporters
- 2010
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Ingår i: FEBS Journal, Special issue: Abstracts of the 35th FEBS Congress, Gothenburg, Sweden, 26 June - 1 July 2010. - 1742-464X. ; 277, supplement 1, s. 209-210
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Glucose plays a central role in nutrient sensing and signalling and its uptake through facilitated diffusion is mediated by membrane associated glucose transporters, GLUT proteins in mammals and Hxt proteins in the yeast S. cerevisiae. These proteins belong to the Major Facilitator Superfamily (MFS), which is present in all studied organisms. They transport a wide range of solutes such as amino acids, sugars, nucleotides, drugs, peptides, organic and inorganic anions, metabolites, neurotransmitters, polyols etc. All MFS proteins structurally investigated have been purified from their natural sources and successful heterologous production has not yet been reported. While there is a wealth of reports in the literature on mutagenesis studies, to analyze the structure-function relationship of glucose transporters, there are no three dimensional structures available. Our research goal is to achieve a more detailed understanding of the structure-function-specificity relationship of glucose transporters. To achieve this overall goal we have successfully expressed mammalian GLUTs and yeast Hxts in yeast. Initial solubilsation trials suggest that Brij35 solubilises GLUT4. Purification and crystallization analyses of the target proteins are ongoing.
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| 3. |
- Eriksson, Stefanie, et al.
(författare)
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NMR quantification of diffusional exchange in cell suspensions with relaxation rate differences between intra and extracellular compartments
- 2017
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:5
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Tidskriftsartikel (refereegranskat)abstract
- Water transport across cell membranes can be measured non-invasively with diffusion NMR. We present a method to quantify the intracellular lifetime of water in cell suspensions with short transverse relaxation times, T2, and also circumvent the confounding effect of different T2 values in the intra- and extracellular compartments. Filter exchange spectroscopy (FEXSY) is specifically sensitive to exchange between compartments with different apparent diffusivities. Our investigation shows that FEXSY could yield significantly biased results if differences in T2 are not accounted for. To mitigate this problem, we propose combining FEXSY with diffusion-relaxation correlation experiment, which can quantify differences in T2 values in compartments with different diffusivities. Our analysis uses a joint constrained fitting of the two datasets and considers the effects of diffusion, relaxation and exchange in both experiments. The method is demonstrated on yeast cells with and without human aquaporins.
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| 4. |
- Hofvander, Jakob, et al.
(författare)
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Frequent low-level mutations of protein kinase D2 in angiolipoma
- 2017
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Ingår i: Journal of Pathology. - : WILEY. - 0022-3417 .- 1096-9896. ; 241:5, s. 578-582
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Tidskriftsartikel (refereegranskat)abstract
- Tumours displaying differentiation towards normal fat constitute the most common subgroup of soft tissue neoplasms. A series of such tumours was investigated by whole-exome sequencing followed by targeted ultra-deep sequencing. Eighty per cent of angiolipomas, but not any other tumour type, displayed mutations in the protein kinase D2 (PRKD2) gene, typically in the part encoding the catalytic domain. The absence of other aberrations at the chromosome or RNA level suggests that PRKD2 mutations are critical for angiolipoma development. Consistently, the mutated PRKD2 alleles were present at low (3-15%) frequencies, indicating that only a subset of the tumour cells is affected. Indeed, by sequencing mature fat cells and other cells separately, the former typically showed the highest mutation frequencies. Thus, we hypothesize that altered PRKD2 signalling in the adipocytic cells drives tumourigenesis and, in agreement with its pivotal role in angiogenesis, induces the vessel formation that is characteristic for angiolipoma. Copyright (c) 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley amp; Sons, Ltd.
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| 5. |
- Nyblom, Anna Maria, 1975, et al.
(författare)
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Exceptional overproduction of a functional human membrane protein
- 2007
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 56:1, s. 110-120
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Tidskriftsartikel (refereegranskat)abstract
- Eukaryotic-especially human-membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQPI in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization. (c) 2007 Elsevier Inc. All rights reserved.
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| 6. |
- Palmgren, Madelene, et al.
(författare)
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Quantification of the Intracellular Life Time of Water Molecules to Measure Transport Rates of Human Aquaglyceroporins
- 2017
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Ingår i: Journal of Membrane Biology. - : Springer Science and Business Media LLC. - 0022-2631 .- 1432-1424. ; 250:6, s. 629-639
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Tidskriftsartikel (refereegranskat)abstract
- Orthodox aquaporins are transmembrane channel proteins that facilitate rapid diffusion of water, while aquaglyceroporins facilitate the diffusion of small uncharged molecules such as glycerol and arsenic trioxide. Aquaglyceroporins play important roles in human physiology, in particular for glycerol metabolism and arsenic detoxification. We have developed a unique system applying the strain of the yeast Pichia pastoris, where the endogenous aquaporins/aquaglyceroporins have been removed and human aquaglyceroporins AQP3, AQP7, and AQP9 are recombinantly expressed enabling comparative permeability measurements between the expressed proteins. Using a newly established Nuclear Magnetic Resonance approach based on measurement of the intracellular life time of water, we propose that human aquaglyceroporins are poor facilitators of water and that the water transport efficiency is similar to that of passive diffusion across native cell membranes. This is distinctly different from glycerol and arsenic trioxide, where high glycerol transport efficiency was recorded.
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| 7. |
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| 8. |
- Rödström, Karin E J, et al.
(författare)
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Two common structural motifs for TCR recognition by staphylococcal enterotoxins
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Superantigens are toxins produced by Staphylococcus aureus, called staphylococcal enterotoxins (abbreviated SEA to SEU). They can cross-link the T cell receptor (TCR) and major histocompatibility complex class II, triggering a massive T cell activation and hence disease. Due to high stability and toxicity, superantigens are potential agents of bioterrorism. Hence, antagonists may not only be useful in the treatment of disease but also serve as countermeasures to biological warfare. Of particular interest are inhibitors against SEA and SEB. SEA is the main cause of food poisoning, while SEB is a common toxin manufactured as a biological weapon. Here, we present the crystal structures of SEA in complex with TCR and SEE in complex with the same TCR, complemented with computational alanine-scanning mutagenesis of SEA, SEB, SEC3, SEE, and SEH. We have identified two common areas that contribute to the general TCR binding for these superantigens. This paves the way for design of single antagonists directed towards multiple toxins.
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| 9. |
- Rödström, Karin, 1986, et al.
(författare)
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Structural studies of staphylococcal enterotoxin H in complex with T cell receptor and major histocompatibility complex class II
- 2010
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Ingår i: FEBS JOURNAL. - 1742-464X. ; 2010, 277, s. 53-54
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Superantigens (SAgs) are bacterial toxins capable of cross-linking the immune receptors of the host, the T cell receptor (TCR) and major histocompatibility complex (MHC) class II, and thereby trigger a massive release of cytokines. This could lead to toxic shock syndrome, which can have a fatal outcome. Here, we present the crystal structure of the ternary complex between the superantigen, staphylococcal enterotoxin H (SEH), TCR and MHC, as well as the dimer complex, including only TCR and SEH. It is evident that SEH interacts with the variable α domain (Vα) of TCR, in sharp contrast to previously studied SAgs that interact with the Vβ domain. Due to the high structural conservation of amino acids in SEH that are crucial for the interaction, we propose that in addition to Vβ activation of T cells, there are SAgs, in addition to SEH, which are able to activate T cells through Vα as well. In addition to providing crucial information regarding the nature of TCR-mediated recognition of superantigens, the finding have central implications for future strategies aimed at preventing or modulating the often pathogenic response to superantigens.
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| 10. |
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