| 1. |
- Akkuratov, Evgeny E.
(författare)
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The Biophysics of Na+,K+-ATPase in neuronal health and disease
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Na+,K+-ATPase is one of the most important proteins in the mammalian cell. It creates sodium and potassium gradients which are fundamental for the membrane potential and sodium-dependent secondary active transport. It has a second role in the cell as a receptor that by binding chemicals from the cardiotonic steroids family, the most knowledgeable of them is ouabain, triggers various signaling pathways in the cell which regulate gene activation, proliferation, apoptosis, etc. It has been shown that several severe neurological diseases are associated with mutations in the Na+,K+-ATPase encoding genes. Although Na+,K+-ATPase was discovered already in 1957 by the Danish scientist Jens Skou, the knowledge about the function of this enzyme is still not complete. In the studies included in the thesis, we have learned more about the function of Na+,K+-ATPase in different aspects of health and disease. In study I we showed a mechanism of ouabain-dependent regulation of the NMDA receptor, one of the most important receptors in the nervous system, via binding with Na+,K+-ATPase. This allows us to look at the Na+,K+-ATPase as regulator via protein-protein interaction. In study II we investigated a different aspect of Na+,K+-ATPase functioning – to look at how binding of ouabain to Na+,K+-ATPase activates a number of signaling cascades by looking at the phosphoproteome status of the cells. This allows us to see the whole picture of ouabain-mediated cascades and further characterize them. In study III we focused on the role of Na+,K+-ATPase in severe epileptic encephalopathy caused by a mutation in the ATP1A1 gene. We performed a molecular and cellular study to describe how mutations affects protein structure and function and found that this mutation converts the ion pump to a nonspecific leak channel. In study IV we performed a translational study of the most common mutation for rapid-onset dystonia-parkinsonism. We studied how this mutation affects the nervous system on the protein-, cellular-, and organism level and found that the complete absence of ultraslow afterhyperpolarization (usAHP) could explain gait disturbances found in patients. In the on-going study we showed that Na+,K+-ATPase can oligomerize and that this effect is triggered by ouabain binding to the Na+,K+-ATPase. In this study, we utilized a novel fluorescence labelling approach and used biophysical techniques with single molecule sensitivity to track Na+,K+-ATPase interactions. In summary, we applied biophysical and molecular methods to study different aspects of the function of Na+,K+-ATPase, and gained insights that could be helpful not only for answering fundamental questions about Na+,K+-ATPase but also to find a treatment for patients with diseases associated with mutations in this protein.
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| 2. |
- Andersson, Magnus, et al.
(författare)
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Transport Pathway in Cu+ P-Type ATPases
- 2014
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 106:2, s. 427A-427A
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 3. |
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| 4. |
- García Pérez, Ana Elia, et al.
(författare)
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Oxygen abundances in metal-poor subgiants as determined from [OI], OI and OH lines
- 2006
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Ingår i: Astronomy and Astrophysics. - : EDP Sciences. - 0004-6361 .- 1432-0746. ; 451:2, s. 621-642
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Tidskriftsartikel (refereegranskat)abstract
- The debate on the oxygen abundances of metal-poor stars has its origin in contradictory results obtained using different abundance indicators. To achieve a better understanding of the problem we have acquired high quality spectra with the Ultraviolet and Visual Echelle Spectrograph at VLT, with a signal-to-noise of the order of 100 in the near ultraviolet and 500 in the optical and near infrared wavelength range. Three different oxygen abundance indicators, OH ultraviolet lines around 310.0 nm, the [O I] line at 630.03 nm and the O I lines at 777.1-5 nm were observed in the spectra of 13 metal-poor subgiants with . Oxygen abundances were obtained from the analysis of these indicators which was carried out assuming local thermodynamic equilibrium and plane-parallel model atmospheres. Abundances derived from O I were corrected for departures from local thermodynamic equilibrium. Stellar parameters were computed using -vs.-color calibrations based on the infrared flux method and Balmer line profiles, Hipparcos parallaxes and lines. [O/Fe] values derived from the forbidden line at 630.03 nm are consistent with an oxygen/iron ratio that varies linearly with [Fe/H] as . Values based on the O I triplet are on average (s.d.) higher than the values based on the forbidden line while the agreement between OH ultraviolet lines and the forbidden line is much better with a mean difference of the order of (s.d.). In general, our results follow the same trend as previously published results with the exception of the ones based on OH ultraviolet lines. In that case our results lie below the values which gave rise to the oxygen abundance debate for metal-poor stars.
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| 5. |
- Han, Huijong, et al.
(författare)
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The XBI BioLab for life science experiments at the European XFEL
- 2021
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Ingår i: Journal of applied crystallography. - 0021-8898 .- 1600-5767. ; 54, s. 7-21
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Tidskriftsartikel (refereegranskat)abstract
- The science of X-ray free-electron lasers (XFELs) critically depends on the performance of the X-ray laser and on the quality of the samples placed into the X-ray beam. The stability of biological samples is limited and key biomolecular transformations occur on short timescales. Experiments in biology require a support laboratory in the immediate vicinity of the beamlines. The XBI BioLab of the European XFEL (XBI denotes XFEL Biology Infrastructure) is an integrated user facility connected to the beamlines for supporting a wide range of biological experiments. The laboratory was financed and built by a collaboration between the European XFEL and the XBI User Consortium, whose members come from Finland, Germany, the Slovak Republic, Sweden and the USA, with observers from Denmark and the Russian Federation. Arranged around a central wet laboratory, the XBI BioLab provides facilities for sample preparation and scoring, laboratories for growing prokaryotic and eukaryotic cells, a Bio Safety Level 2 laboratory, sample purification and characterization facilities, a crystallization laboratory, an anaerobic laboratory, an aerosol laboratory, a vacuum laboratory for injector tests, and laboratories for optical microscopy, atomic force microscopy and electron microscopy. Here, an overview of the XBI facility is given and some of the results of the first user experiments are highlighted.
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| 6. |
- Hjorth-Jensen, Samuel John, et al.
(författare)
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Prospects for membrane protein crystals in NMX
- 2020
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Ingår i: Neutron Crystallography in Structural Biology. - : Elsevier. - 1557-7988 .- 0076-6879. ; 634, s. 47-68
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Bokkapitel (refereegranskat)abstract
- Adding hydrogen atoms and protonation states to structures of membrane proteins requires successful implementation of neutron macromolecular crystallography (NMX). This information would significantly increase our fundamental understanding of the transport processes membrane proteins undertake. To grow the large crystals needed for NMX studies requires significant amounts of stable protein, but once that challenge is overcome there is no intrinsic property of membrane proteins preventing the growth of large crystals per se. The calcium-transporting P-type ATPase (SERCA) has been thoroughly characterized biochemically and structurally over decades. We have extended our crystallization efforts to assess the feasibility of growing SERCA crystals for NMX—exploring microdialysis and capillary counterdiffusion crystallization techniques as alternatives to the traditional vapor diffusion crystallization experiment. Both methods possess crystallization dynamics favorable for maximizing crystal size and we used them to facilitate the growth of large crystals, validating these approaches for membrane protein crystallization for NMX.
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| 7. |
- Jonsell, Karin, et al.
(författare)
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Chemical abundances in 43 metal-poor stars
- 2005
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Ingår i: Astronomy & Astrophysics. - : EDP Sciences. - 0004-6361 .- 1432-0746. ; 440:1, s. 321-343
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Tidskriftsartikel (refereegranskat)abstract
- We have derived abundances of O, Na, Mg, Al, Si, Ca, Sc, Ti, V, Cr, Fe, Ni, and Ba for 43 metal-poor field stars in the solar neighbourhood, most of them subgiants or turn-off-point stars with iron abundances [Fe/H] ranging from -0.4 to -3.0. About half of this sample has not been spectroscopically analysed in detail before. Effective temperatures were estimated from uvby photometry, and surface gravities primarily from Hipparcos parallaxes. The analysis is differential relative to the Sun, and was carried out with plane-parallel MARCS models. Various sources of error are discussed and found to contribute a total error of about 0.1-0.2 dex for most elements, while relative abundances, such as [Ca/Fe], are most probably more accurate. For the oxygen abundances, determined in an NLTE analysis of the 7774 Å triplet lines, the errors may be somewhat larger. We made a detailed comparison with similar studies and traced the reasons for the, in most cases, relatively small differences. Among the results we find that [O/Fe] possibly increases beyond [Fe/H] = -1.0, though considerably less so than in results obtained by others from abundances based on OH lines. We did not trace any tendency toward strong overionization of iron, and find the excesses, relative to Fe and the Sun, of the α elements Mg, Si, and Ca to be smaller than those of O. We discuss some indications that also the abundances of different α elements relative to Fe vary and the possibility that some of the scatter around the trends in abundances relative to iron may be real. This may support the idea that the formation of Halo stars occurred in smaller systems with different star formation rates. We verify the finding by Gratton et al. (2003b, A&A, 406, 131) that stars that do not participate in the rotation of the galactic disk show a lower mean and larger spread in [ α/Fe] than stars participating in the general rotation. The latter stars also seem to show some correlation between [ α/Fe] and rotation speed. We trace some stars with peculiar abundances, among these two Ba stars, HD 17072 and HD 196944, the second already known to be rich in s elements. Finally we advocate that a spectroscopic study of a larger sample of halo stars with well-defined selection criteria is very important, in order to add to the very considerable efforts that various groups have already made.
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| 8. |
- Kirscht, Andreas, et al.
(författare)
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Crystal Structure of an Ammonia-Permeable Aquaporin
- 2016
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Ingår i: PLoS Biology. - : Public Library of Science (PLoS). - 1545-7885. ; 14:3
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporins of the TIP subfamily (Tonoplast Intrinsic Proteins) have been suggested to facilitate permeation of water and ammonia across the vacuolar membrane of plants, allowing the vacuole to efficiently sequester ammonium ions and counteract cytosolic fluctuations of ammonia. Here, we report the structure determined at 1.18 Å resolution from twinned crystals of Arabidopsis thaliana aquaporin AtTIP2;1 and confirm water and ammonia permeability of the purified protein reconstituted in proteoliposomes as further substantiated by molecular dynamics simulations. The structure of AtTIP2;1 reveals an extended selectivity filter with the conserved arginine of the filter adopting a unique unpredicted position. The relatively wide pore and the polar nature of the selectivity filter clarify the ammonia permeability. By mutational studies, we show that the identified determinants in the extended selectivity filter region are sufficient to convert a strictly water-specific human aquaporin into an AtTIP2;1-like ammonia channel. A flexible histidine and a novel water-filled side pore are speculated to deprotonate ammonium ions, thereby possibly increasing permeation of ammonia. The molecular understanding of how aquaporins facilitate ammonia flux across membranes could potentially be used to modulate ammonia losses over the plasma membrane to the atmosphere, e.g., during photorespiration, and thereby to modify the nitrogen use efficiency of plants.
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| 9. |
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| 10. |
- Mattle, Daniel, et al.
(författare)
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A sulfur-based transport pathway in Cu+-ATPases
- 2015
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Ingår i: EMBO Reports. - : EMBO. - 1469-221X .- 1469-3178. ; 16:6, s. 40-728
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Tidskriftsartikel (refereegranskat)abstract
- Cells regulate copper levels tightly to balance the biogenesis and integrity of copper centers in vital enzymes against toxic levels of copper. PIB -type Cu(+)-ATPases play a central role in copper homeostasis by catalyzing the selective translocation of Cu(+) across cellular membranes. Crystal structures of a copper-free Cu(+)-ATPase are available, but the mechanism of Cu(+) recognition, binding, and translocation remains elusive. Through X-ray absorption spectroscopy, ATPase activity assays, and charge transfer measurements on solid-supported membranes using wild-type and mutant forms of the Legionella pneumophila Cu(+)-ATPase (LpCopA), we identify a sulfur-lined metal transport pathway. Structural analysis indicates that Cu(+) is bound at a high-affinity transmembrane-binding site in a trigonal-planar coordination with the Cys residues of the conserved CPC motif of transmembrane segment 4 (C382 and C384) and the conserved Met residue of transmembrane segment 6 (M717 of the MXXXS motif). These residues are also essential for transport. Additionally, the studies indicate essential roles of other conserved intramembranous polar residues in facilitating copper binding to the high-affinity site and subsequent release through the exit pathway.
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| 11. |
- Nass, Karol, et al.
(författare)
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Indications of radiation damage in ferredoxin microcrystals using high-intensity X-FEL beams
- 2015
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Ingår i: Journal of Synchrotron Radiation. - 0909-0495 .- 1600-5775. ; 22:2, s. 225-238
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Tidskriftsartikel (refereegranskat)abstract
- Proteins that contain metal cofactors are expected to be highly radiation sensitive since the degree of X-ray absorption correlates with the presence of high-atomic-number elements and X-ray energy. To explore the effects of local damage in serial femtosecond crystallography (SFX), Clostridium ferredoxin was used as a model system. The protein contains two [4Fe–4S] clusters that serve as sensitive probes for radiation-induced electronic and structural changes. High-dose room-temperature SFX datasets were collected at the Linac Coherent Light Source of ferredoxin microcrystals. Difference electron density maps calculated from high-dose SFX and synchrotron data show peaks at the iron positions of the clusters, indicative of decrease of atomic scattering factors due to ionization. The electron density of the two [4Fe–4S] clusters differs in the FEL data, but not in the synchrotron data. Since the clusters differ in their detailed architecture, this observation is suggestive of an influence of the molecular bonding and geometry on the atomic displacement dynamics following initial photoionization. The experiments are complemented by plasma code calculations.
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| 12. |
- Nissen, Poul Erik, et al.
(författare)
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High-precision stellar abundances of the elements : methods and applications
- 2018
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Ingår i: The Astronomy and Astrophysics Review. - : SPRINGER. - 0935-4956 .- 1432-0754. ; 26
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Tidskriftsartikel (refereegranskat)abstract
- Efficient spectrographs at large telescopes have made it possible to obtain high-resolution spectra of stars with high signal-to-noise ratio and advances in model atmosphere analyses have enabled estimates of high-precision differential abundances of the elements from these spectra, i.e. with errors in the range 0.01-0.03dex for F, G, and K stars. Methods to determine such high-precision abundances together with precise values of effective temperatures and surface gravities from equivalent widths of spectral lines or by spectrum synthesis techniques are outlined, and effects on abundance determinations from using a 3D non-LTE analysis instead of a classical 1D LTE analysis are considered. The determination of high-precision stellar abundances of the elements has led to the discovery of unexpected phenomena and relations with important bearings on the astrophysics of galaxies, stars, and planets, i.e. (i) Existence of discrete stellar populations within each of the main Galactic components (disk, halo, and bulge) providing new constraints on models for the formation of the Milky Way. (ii) Differences in the relation between abundances and elemental condensation temperature for the Sun and solar twins suggesting dust-cleansing effects in proto-planetary disks and/or engulfment of planets by stars; (iii) Differences in chemical composition between binary star components and between members of open or globular clusters showing that star- and cluster-formation processes are more complicated than previously thought; (iv) Tight relations between some abundance ratios and age for solar-like stars providing new constraints on nucleosynthesis and Galactic chemical evolution models as well as the composition of terrestrial exoplanets. We conclude that if stellar abundances with precisions of 0.01-0.03dex can be achieved in studies of more distant stars and stars on the giant and supergiant branches, many more interesting future applications, of great relevance to stellar and galaxy evolution, are probable. Hence, in planning abundance surveys, it is important to carefully balance the need for large samples of stars against the spectral resolution and signal-to-noise ratio needed to obtain high-precision abundances. Furthermore, it is an advantage to work differentially on stars with similar atmospheric parameters, because then a simple 1D LTE analysis of stellar spectra may be sufficient. However, when determining high-precision absolute abundances or differential abundance between stars having more widely different parameters, e.g. metal-poor stars compared to the Sun or giants to dwarfs, then 3D non-LTE effects must be taken into account.
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| 13. |
- Nyblom, Maria, et al.
(författare)
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Crystal Structure of Na+, K+-ATPase in the Na+-Bound State
- 2013
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 342:6154, s. 123-127
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Tidskriftsartikel (refereegranskat)abstract
- The Na+, K+-adenosine triphosphatase (ATPase) maintains the electrochemical gradients of Na+ and K+ across the plasma membrane-a prerequisite for electrical excitability and secondary transport. Hitherto, structural information has been limited to K+-bound or ouabain-blocked forms. We present the crystal structure of a Na+-bound Na+, K+-ATPase as determined at 4.3 angstrom resolution. Compared with the K+-bound form, large conformational changes are observed in the a subunit whereas the beta and gamma subunit structures are maintained. The locations of the three Na+ sites are indicated with the unique site III at the recently suggested IIIb, as further supported by electrophysiological studies on leak currents. Extracellular release of the third Na+ from IIIb through IIIa, followed by exchange of Na+ for K+ at sites I and II, is suggested.
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| 14. |
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| 15. |
- Paulsen, Peter Aasted, et al.
(författare)
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The C-terminal cavity of the Na,K-ATPase analyzed by docking and electrophysiology
- 2013
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Ingår i: Molecular membrane biology. - : Informa UK Limited. - 0968-7688 .- 1464-5203. ; 30:2, s. 195-205
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Tidskriftsartikel (refereegranskat)abstract
- The Na,K-ATPase is essential to all animals, since it maintains the electrochemical gradients that energize the plasma membrane. Naturally occurring inhibitors of the pump from plants have been used pharmaceutically in cardiac treatment for centuries. The inhibitors block the pump by binding on its extracellular side and thereby locking it. To explore the possibilities for designing an alternative way of targeting the pump function, we have examined the structural requirements for binding to a pocket that accommodates the two C-terminal residues, YY, in the crystal structures of the pump. To cover the sample space of two residues, we first performed docking studies with the 400 possible dipeptides. For validation of the in silico predictions, pumps with 13 dipeptide sequences replacing the C-terminal YY were expressed in Xenopus laevis oocytes and examined with electrophysiology. Our data show a significant correlation between the docking scores from two different methods and the experimentally determined sodium affinities, which strengthens the previous hypothesis that sodium binding is coupled to docking of the C-terminus. From the dipeptides that dock the best and better than wild-type YY, it may therefore be possible to develop specific drugs targeting a previously unexplored binding pocket in the sodium pump.
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| 16. |
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| 17. |
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| 18. |
- Ravishankar, Harsha, et al.
(författare)
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Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering
- 2020
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Ingår i: Science Advances. - : American Association for the Advancement of Science. - 2375-2548. ; 6:12
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Tidskriftsartikel (refereegranskat)abstract
- Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca-2] E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment.
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| 19. |
- Sitsel, Oleg, et al.
(författare)
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Structure and Function of Cu(I)- and Zn(II)-ATPases
- 2015
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 54:37, s. 5673-5683
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Tidskriftsartikel (refereegranskat)abstract
- Copper and zinc are micronutrients essential for the function of many enzymes while also being toxic at elevated concentrations. Cu(I)- and Zn(II)-transporting P-type ATPases of subclass 1B are of key importance for the homeostasis of these transition metals, allowing ion transport across cellular membranes at the expense of ATP. Recent biochemical studies and crystal structures have significantly improved our understanding of the transport mechanisms of these proteins, but many details about their structure and function remain elusive. Here we compare the Cu(I)- and Zn(II)-ATPases, scrutinizing the molecular differences that allow transport of these two distinct metal types, and discuss possible future directions of research in the field.
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| 20. |
- Skjørringe, Tina, et al.
(författare)
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Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Menkes disease (MD) is caused by mutations in ATP7A, encoding a copper-transporting P-type ATPase which exhibits copper-dependent trafficking. ATP7A is found in the Trans-Golgi Network (TGN) at low copper concentrations, and in the post-Golgi compartments and the plasma membrane at higher concentrations. Here we have analyzed the effect of 36 ATP7A missense mutations identified in phenotypically different MD patients. Nine mutations identified in patients with severe MD, virtually eliminated ATP7A synthesis, in most cases due to aberrant RNA splicing. A group of 21 predominantly severe mutations led to trapping of the protein in TGN and displayed essentially no activity in a yeast-based functional assay. These were predicted to inhibit the catalytic phosphorylation of the protein. Four mutants showed diffuse post-TGN localization, while two displayed copper dependent trafficking. These six variants were identified in patients with mild MD and typically displayed activity in the yeast assay. The four post-TGN located mutants were presumably affected in the catalytic dephosphorylation of the protein. Together these results indicate that the severity of MD correlate with cellular localization of ATP7A and support previous studies indicating that phosphorylation is crucial for the exit of ATP7A from TGN, while dephosphorylation is crucial for recycling back to TGN.
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| 21. |
- Sørensen, Thomas Lykke Møller, et al.
(författare)
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Membrane-protein crystals for neutron diffraction
- 2018
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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 74:12, s. 1208-1218
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Forskningsöversikt (refereegranskat)abstract
- Neutron macromolecular crystallography (NMX) has the potential to provide the experimental input to address unresolved aspects of transport mechanisms and protonation in membrane proteins. However, despite this clear scientific motivation, the practical challenges of obtaining crystals that are large enough to make NMX feasible have so far been prohibitive. Here, the potential impact on feasibility of a more powerful neutron source is reviewed and a strategy for obtaining larger crystals is formulated, exemplified by the calcium-transporting ATPase SERCA1. The challenges encountered at the various steps in the process from crystal nucleation and growth to crystal mounting are explored, and it is demonstrated that NMX-compatible membrane-protein crystals can indeed be obtained.
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| 22. |
- Villa, Elizabeth, et al.
(författare)
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Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis
- 2009
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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. - 0027-8424 .- 1091-6490. ; 106:4, s. 1063-1068
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Tidskriftsartikel (refereegranskat)abstract
- In translation, elongation factor Tu (EF-Tu) molecules deliver aminoacyl-tRNAs to the mRNA-programmed ribosome. The GTPase activity of EF-Tu is triggered by ribosome-induced conformational changes of the factor that play a pivotal role in the selection of the cognate aminoacyl-tRNAs. We present a 6.7-A cryo-electron microscopy map of the aminoacyl-tRNA x EF-Tu x GDP x kirromycin-bound Escherichia coli ribosome, together with an atomic model of the complex obtained through molecular dynamics flexible fitting. The model reveals the conformational changes in the conserved GTPase switch regions of EF-Tu that trigger hydrolysis of GTP, along with key interactions, including those between the sarcin-ricin loop and the P loop of EF-Tu, and between the effector loop of EF-Tu and a conserved region of the 16S rRNA. Our data suggest that GTP hydrolysis on EF-Tu is controlled through a hydrophobic gate mechanism.
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| 23. |
- Wang, Kaituo, et al.
(författare)
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Structure and mechanism of Zn2+-transporting P-type ATPases
- 2014
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 514:7523, s. 518-
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Tidskriftsartikel (refereegranskat)abstract
- Zinc is an essential micronutrient for all living organisms. It is required for signalling and proper functioning of a range of proteins involved in, for example, DNA binding and enzymatic catalysis(1). In prokaryotes and photosynthetic eukaryotes, Zn2+-transporting P-type ATPases of class IB (ZntA) are crucial for cellular redistribution and detoxification of Zn2+ and related elements(2,3). Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2.P-i) of ZntA from Shigella sonnei, determined at 3.2 angstrom and 2.7 angstrom resolution, respectively. The structures reveal a similar fold to Cu+-ATPases, with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn2+ ions by the transporter. The E2P structure displays a wide extracellular release pathway reaching the invariant residues at the high-affinity site, including C392, C394 and D714. The pathway closes in the E2.P-i state, in which D714 interacts with the conserved residue K693, which possibly stimulates Zn2+ release as a built-in counter ion, as has been proposed for H+-ATPases. Indeed, transport studies in liposomes provide experimental support for ZntA activity without counter transport. These findings suggest a mechanistic link between P-IB-type Zn2+-ATPases and P-III-type H+-ATPases and at the same time show structural features of the extracellular release pathway that resemble P-II-type ATPases such as the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase(4,5) (SERCA) and Na+, K+-ATPase(6). These findings considerably increase our understanding of zinc transport in cells and represent new possibilities for biotechnology and biomedicine.
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