| 1. |
- Adawy, Alaa, et al.
(författare)
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High Resolution Protein Crystals Using an Efficient Convection-Free Geometry
- 2013
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Ingår i: Crystal Growth & Design. - : American Chemical Society (ACS). - 1528-7483 .- 1528-7505. ; 13:2, s. 775-781
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Tidskriftsartikel (refereegranskat)abstract
- Macromolecular crystallography is the most direct and accurate approach to determine the three-dimensional structure of biological macromolecules. The growth of high quality single crystals, yielding diffraction to the highest X-ray resolution, remains a bottleneck in this methodology. Here we show that through a modification of the batch crystallization method, an entirely convection-free crystallization environment is achieved, which enhances the purity and crystallinity of protein crystals. This is accomplished by using an upside-down geometry, where crystals grow at the “ceiling” of a growth-cell completely filled with the crystallization solution. The “ceiling crystals” experience the same diffusion-limited conditions as in space microgravity experiments. The new method was tested on bovine insulin and two hen egg-white lysozyme polymorphs. In all cases, ceiling crystals diffracted X-rays to resolution limits beyond that for other methods using similar crystallization conditions without further optimization. In addition, we demonstrate that the ceiling crystallization method leads to crystals with much lower impurity incorporation.
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| 2. |
- Frick, Anna, 1982, et al.
(författare)
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Mercury increases water permeability of a plant aquaporin through a non-cysteine-related mechanism
- 2013
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 454:pt 3, s. 491-499
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Tidskriftsartikel (refereegranskat)abstract
- Water transport across cellular membranes is mediated by a family of membrane proteins known as AQPs (aquaporins). AQPs were first discovered on the basis of their ability to be inhibited by mercurial compounds, an experiment which has followed the AQP field ever since. Although mercury inhibition is most common, many AQPs are mercury insensitive. In plants, regulation of AQPs is important in order to cope with environmental changes. Plant plasma membrane AQPs are known to be gated by phosphorylation, pH and Ca2+. We have previously solved the structure of the spinach AQP SoPIP2;1 (Spinacia oleracea plasma membrane intrinsic protein 2; 1) in closed and open conformations and proposed a mechanism for how this gating can be achieved. To study the effect of mercury on SoPIP2; 1 we solved the structure of the SoPIP2;1-mercury complex and characterized the water transport ability using proteoliposomes. The structure revealed mercury binding to three out of four cysteine residues. In contrast to what is normally seen for AQPs, mercury increased the water transport rate of SoPIP2; 1, an effect which could not be attributed to any of the cysteine residues. This indicates that other factors might influence the effect of mercury on SoPIP2; 1, one of which could be the properties of the lipid bilayer.
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| 3. |
- Frick, Anna, 1982, et al.
(författare)
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Structural basis for pH gating of plant aquaporins
- 2013
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Ingår i: Febs Letters. - : Wiley. - 0014-5793. ; 587:7, s. 989-993
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Tidskriftsartikel (refereegranskat)abstract
- Plants have evolved to cope with fluctuations in water supply by gating their water channels known
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| 4. |
- Frick, Anna, 1982, et al.
(författare)
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X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking.
- 2014
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 111:17, s. 6305-10
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Tidskriftsartikel (refereegranskat)abstract
- Human aquaporin 2 (AQP2) is a water channel found in the kidney collecting duct, where it plays a key role in concentrating urine. Water reabsorption is regulated by AQP2 trafficking between intracellular storage vesicles and the apical membrane. This process is tightly controlled by the pituitary hormone arginine vasopressin and defective trafficking results in nephrogenic diabetes insipidus (NDI). Here we present the X-ray structure of human AQP2 at 2.75 Å resolution. The C terminus of AQP2 displays multiple conformations with the C-terminal α-helix of one protomer interacting with the cytoplasmic surface of a symmetry-related AQP2 molecule, suggesting potential protein-protein interactions involved in cellular sorting of AQP2. Two Cd(2+)-ion binding sites are observed within the AQP2 tetramer, inducing a rearrangement of loop D, which facilitates this interaction. The locations of several NDI-causing mutations can be observed in the AQP2 structure, primarily situated within transmembrane domains and the majority of which cause misfolding and ER retention. These observations provide a framework for understanding why mutations in AQP2 cause NDI as well as structural insights into AQP2 interactions that may govern its trafficking.
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| 5. |
- Nilsson, Daniel
(författare)
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Insane in the membrane: : Insertion of marginally hyrdophobic transmembrane helices and global analysis of membrane protein topology
- 2014
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are responsible for carrying out most of the tasks in a living cell; transcription, translation, replication, movement, catalysis and communication to mention a few. A subset of proteins is the integral membrane proteins, IMPs, which play an important role in governing communication across membranes, whether it is the plasma membrane or an organelle membrane. As IMPs are often responsible for uptake of substances such as hormones and pharmaceuticals they are also very often prime targets in medical research. In order for an IMP to function correctly it must fold and insert into the membrane properly as well as display the correct motifs to its surroundings, on either side of the membrane. IMPs cannot spontaneously insert into the membrane, the insertion is assisted by the Sec-translocon machinery. The Sec-translocon creates a pore in the membrane enabling nascent polypeptides to translocate across the membrane. This thesis will cover both the insertion of IMP segments as well as an evaluation of different approaches on how to investigate the topology of integral membrane proteins.The first question to be addressed is whether there are any specific sequence motifs within the sequence context that can improve the co-translational insertion of a marginally hydrophobic transmembrane helix, mTMH, into the membrane. A mTMH is a protein segment that would not insert by itself into the membrane. It has however been shown that these mTMHs can insert effectively into the membrane using their neighboring helices and loops, referred to as its sequence context, to compensate for the unfavorable insertion of only the mTMH. We show for a number of mTMHs that disrupting the sequence context motifs, usually lowering the ΔG-values for insertion by introducing more hydrophobic residues through substitutions in the sequence context, does not by itself improve the insertion of a given mTMH. It can however be concluded that the positive inside rule is of great importance to improve recognition and co-translational insertion of these mTMHs as it provides an oriental preference of the subsequent helix. This oriental preference will enable the mTMH to insert. This means that the positive inside rule it stronger when followed by a transmembrane helix, at least for the insertion of mTMHs.The second question addressed is that of how to design a method to analyze the topology of membrane proteins in a high-throughput proteomic fashion. In order to extract information on membrane protein topology a protease can be used to degrade the exposed parts of the integral membrane protein, known as shaving. These peptides can then subsequently be degraded and analyzed using MS and bioinformatics. To compare different proteases, we first apply our shaving experiment on two over-expressed proteins and analyze the detected peptides using MS. Secondly; we run the same experiment on non-over-expressed Escherichia coli membrane proteins with known structure. Finally, the results from the above experiments were used to test the accuracy of a number of topology predictors. We can conclude that the use of the protease Thermolysin does show promising results when compared to for instance trypsin. Even though the two proteases show somewhat similar output on the proteins used in this study, Thermolysin does produce fewer peptides originating from the transmembrane region. This is most likely due to the milder, more native like reaction conditions combined with the shorter incubation time used for Thermolysin as compared to trypsin. These properties are believed to greatly improve the output and accuracy when applied on large scale global analysis.
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| 6. |
- Törnroth-Horsefield, Susanna, 1973, et al.
(författare)
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Structural insights into eukaryotic aquaporin regulation.
- 2010
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Ingår i: FEBS letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 584:12, s. 2580-8
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporin-mediated water transport across cellular membranes is an ancient, ubiquitous mechanism within cell biology. This family of integral membrane proteins includes both water selective pores (aquaporins) and transport facilitators of other small molecules such as glycerol and urea (aquaglyceroporins). Eukaryotic aquaporins are frequently regulated post-translationally by gating, whereby the rate of flux through the channel is controlled, or by trafficking, whereby aquaporins are shuttled from intracellular storage sites to the plasma membrane. A number of high-resolution X-ray structures of eukaryotic aquaporins have recently been reported and the new structural insights into gating and trafficking that emerged from these studies are described. Basic structural themes reoccur, illustrating how the problem of regulation in diverse biological contexts builds upon a limited set of possible solutions.
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