| 1. |
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| 2. |
- Andersson, Rebecka, 1988, et al.
(författare)
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Serial femtosecond crystallography structure of cytochrome c oxidase at room temperature.
- 2017
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Cytochrome c oxidase catalyses the reduction of molecular oxygen to water while the energy released in this process is used to pump protons across a biological membrane. Although an extremely well-studied biological system, the molecular mechanism of proton pumping by cytochrome c oxidase is still not understood. Here we report a method to produce large quantities of highly diffracting microcrystals of ba 3-type cytochrome c oxidase from Thermus thermophilus suitable for serial femtosecond crystallography. The room-temperature structure of cytochrome c oxidase is solved to 2.3Å resolution from data collected at an X-ray Free Electron Laser. We find overall agreement with earlier X-ray structures solved from diffraction data collected at cryogenic temperature. Previous structures solved from synchrotron radiation data, however, have shown conflicting results regarding the identity of the active-site ligand. Our room-temperature structure, which is free from the effects of radiation damage, reveals that a single-oxygen species in the form of a water molecule or hydroxide ion is bound in the active site. Structural differences between the ba 3-type and aa 3-type cytochrome c oxidases around the proton-loading site are also described.
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| 3. |
- Andersson, Rebecka, 1988, et al.
(författare)
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Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments.
- 2019
-
Ingår i: Acta crystallographica. Section D, Structural biology. - 2059-7983. ; 75:Pt 10, s. 937-946
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Tidskriftsartikel (refereegranskat)abstract
- Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.
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| 4. |
- Brändén, Gisela, 1975, et al.
(författare)
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Advances and challenges in time-resolved macromolecular crystallography.
- 2021
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 373:6558
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Forskningsöversikt (refereegranskat)abstract
- Conformational changes within biological macromolecules control a vast array of chemical reactions in living cells. Time-resolved crystallography can reveal time-dependent structural changes that occur within protein crystals, yielding chemical insights in unparalleled detail. Serial crystallography approaches developed at x-ray free-electron lasers are now routinely used for time-resolved diffraction studies of macromolecules. These techniques are increasingly being applied at synchrotron radiation sources and to a growing diversity of macromolecules. Here, we review recent progress in the field, including visualizing ultrafast structural changes that guide the initial trajectories of light-driven reactions as well as capturing biologically important conformational changes on slower time scales, for which bacteriorhodopsin and photosystem II are presented as illustrative case studies.
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| 5. |
- Brändén, Gisela, 1975, et al.
(författare)
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Coherent diffractive imaging of microtubules using an X-ray laser.
- 2019
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10:1
-
Tidskriftsartikel (refereegranskat)abstract
- X-ray free electron lasers (XFELs) create new possibilities for structural studies of biological objects that extend beyond what is possible with synchrotron radiation. Serial femtosecond crystallography has allowed high-resolution structures to be determined from micro-meter sized crystals, whereas single particle coherent X-ray imaging requires development to extend the resolution beyond a few tens of nanometers. Here we describe an intermediate approach: the XFEL imaging of biological assemblies with helical symmetry. We collected X-ray scattering images from samples of microtubules injected across an XFEL beam using a liquid microjet, sorted these images into class averages, merged these data into a diffraction pattern extending to 2nm resolution, and reconstructed these data into a projection image of the microtubule. Details such as the 4nm tubulin monomer became visible in this reconstruction. These results illustrate the potential of single-molecule X-ray imaging of biological assembles with helical symmetry at room temperature.
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| 6. |
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| 7. |
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| 8. |
- Brändén, Gisela, 1975-
(författare)
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Structure and Function of Cytochrome c Oxidase
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Aerobic organisms, ranging from bacteria to humans, obtain their energy through the process of respiration. Electrons from the breakdown of food are transported through the membrane-bound enzyme complexes of the respiratory chain, to the terminal acceptor molecule, oxygen. The energy released in the process is used to transport protons across the cell membrane, thereby establishing an electrochemical gradient that can be utilised e.g. to synthesise ATP. The last respiratory complex in mitochondria and many bacteria is cytochrome c oxidase (CcO). CcO catalyses the reduction of oxygen to water, and at the same time pumps protons from the more negative to the more positive side of the membrane. In spite of extended and intensive research efforts, the detailed mechanism by which the exergonic electron-transfer reactions are coupled to the endergonic proton pumping is still not understood.The focus of this thesis is the structure and function of CcO from the bacterium Rhodobacter sphaeroides. The structures of the wild-type enzyme and two mutant enzymes have been investigated, providing insights into structural changes taking place upon replacing a glutamate residue (E286) in one of the proton-transfer pathways. E286 is a key element in directing protons either to the catalytic site for the oxygen chemistry or towards the outside for pumping. Results from time-resolved optical absorption spectroscopy show that the pKa of E286 has to be close to 9.5 for the proton pump to function, and that the pKa can be modulated by mutations at a distance of ~25 Å from E286. From another study we conclude that the D-propionate of haem a3 is most likely the acceptor site for pumped protons. Finally, we have identified the structural element which conformational changes determine the reaction rate of each proton-pumping step.The results are summarised in a molecular model for proton pumping by CcO, in which deprotonation of E286 drives proton translocation across the membrane.
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| 9. |
- Brändén, Gisela, 1975, et al.
(författare)
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Structure-based ligand design to overcome CYP inhibition in drug discovery projects
- 2014
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Ingår i: Drug Discovery Today. - : Elsevier BV. - 1359-6446. ; 19:7, s. 905-911
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Forskningsöversikt (refereegranskat)abstract
- Cytochrome P450 (CYP) enzymes are key players in xenobiotic metabolism, and inhibition of CYPs can therefore result in unwanted drug-drug interactions. Within drug discovery, CYP inhibition can cause delays in the progression of candidate drugs, or even premature closure of projects. During the past decade, a massive effort in the pharmaceutical industry and academic research has produced a wealth of structural information in the CYP field. In this short review, we will describe how structure-based approaches can be used in the pharmaceutical industry to work away from CYP inhibition, with a focus on the opportunities and challenges. We will show two examples from our own work where structural information on CYP2C9 and CYP3A4 inhibitor complexes have been successfully exploited in ongoing drug discovery projects.
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| 10. |
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| 11. |
- Busenlehner, Laura, et al.
(författare)
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Structural Elements Involved in Proton Translocation by Cytochrome c Oxidase as Revealed by Backbone Amide Hydrogen-Deuterium Exchange of the E286H Mutant
- 2008
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Ingår i: Biochemistry. - 0006-2960 .- 1520-4995. ; 47:1, s. 73-83
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Tidskriftsartikel (refereegranskat)abstract
- Cytochrome c oxidase is the terminal electron acceptor in the respiratory chains of aerobic organisms and energetically couples' the reduction of oxygen to water to proton pumping across the membrane. The mechanisms of proton uptake, gating, and pumping have yet to be completely elucidated at the molecular level for these enzymes. For Rhodobacter sphaeroides CytcO (cytochrome aa3), it appears as though the E286 side chain of subunit I is a branching point from which protons are shuttled either to the catalytic site for O2 reduction or to the acceptor site for pumped protons. Amide hydrogen-deuterium exchange mass spectrometry was used to investigate how mutation of this key branching residue to histidine (E286H) affects the structures and dynamics of four redox intermediate states. A functional characterization of this mutant reveals that E286H CytcO retains ∼1% steady-state activity that is uncoupled from proton pumping and that proton transfer from H286 is significantly slowed. Backbone amide H-D exchange kinetics indicates that specific regions of CytcO, perturbed by the E286H mutation, are likely to be involved in proton gating and in the exit pathway for pumped protons. The results indicate that redox-dependent conformational changes around E286 are essential for internal proton transfer. E286H CytcO, however, is incapable of these specific conformational changes and therefore is insensitive to the redox state of the enzyme. These data support a model where the side chain conformation of E286 controls proton translocation in CytcO through its interactions with the proton gate, which directs the flow of protons either to the active site or to the exit pathway. In the E286H mutant, the proton gate does not function properly and the exit channel is unresponsive. These results provide new insight into the structure and mechanism of proton transtocation by CytcO.
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| 12. |
- Båth, Petra, 1988, et al.
(författare)
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Lipidic cubic phase serial femtosecond crystallography structure of a photosynthetic reaction centre
- 2022
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Ingår i: Acta Crystallographica Section D-Structural Biology. - : International Union of Crystallography (IUCr). - 2059-7983. ; 78, s. 698-708
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Tidskriftsartikel (refereegranskat)abstract
- Serial crystallography is a rapidly growing method that can yield structural insights from microcrystals that were previously considered to be too small to be useful in conventional X-ray crystallography. Here, conditions for growing microcrystals of the photosynthetic reaction centre of Blastochloris viridis within a lipidic cubic phase (LCP) crystallization matrix that employ a seeding protocol utilizing detergent-grown crystals with a different crystal packing are described. LCP microcrystals diffracted to 2.25 angstrom resolution when exposed to XFEL radiation, which is an improvement of 0.15 angstrom over previous microcrystal forms. Ubiquinone was incorporated into the LCP crystallization media and the resulting electron density within the mobile Q(B) pocket is comparable to that of other cofactors within the structure. As such, LCP microcrystallization conditions will facilitate time-resolved diffraction studies of electron-transfer reactions to the mobile quinone, potentially allowing the observation of structural changes associated with the two electron-transfer reactions leading to complete reduction of the ubiquinone ligand.
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| 13. |
- Dods, Robert, 1989, et al.
(författare)
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From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography.
- 2017
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Ingår i: Structure. - : Elsevier BV. - 1878-4186 .- 0969-2126. ; 25:9, s. 1461-1468
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Tidskriftsartikel (refereegranskat)abstract
- Serial protein crystallography was developed at X-ray free-electron lasers (XFELs) and is now also being applied at storage ring facilities. Robust strategies for the growth and optimization of microcrystals are needed to advance the field. Here we illustrate a generic strategy for recovering high-density homogeneous samples of microcrystals starting from conditions known to yield large (macro) crystals of the photosynthetic reaction center of Blastochloris viridis (RCvir). We first crushed these crystals prior to multiple rounds of microseeding. Each cycle of microseeding facilitated improvements in the RCvir serial femtosecond crystallography (SFX) structure from 3.3-Å to 2.4-Å resolution. This approach may allow known crystallization conditions for other proteins to be adapted to exploit novel scientific opportunities created by serial crystallography.
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| 14. |
- Dods, Robert, 1989, et al.
(författare)
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Ultrafast structural changes within a photosynthetic reaction centre.
- 2021
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 589:7841, s. 310-314
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Tidskriftsartikel (refereegranskat)abstract
- Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography1 using an X-ray free-electron laser2 to observe light-induced structural changes in the photosynthetic reaction centre of Blastochloris viridis on a timescale of picoseconds. Structural perturbations first occur at the special pair of chlorophyll molecules of the photosynthetic reaction centre that are photo-oxidized by light. Electron transfer to the menaquinone acceptor on the opposite side of the membrane induces a movement of this cofactor together with lower amplitude protein rearrangements. These observations reveal how proteins use conformational dynamics to stabilize the charge-separation steps of electron-transfer reactions.
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| 15. |
- Ghosh, Swagatha, 1987, et al.
(författare)
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A simple goniometer-compatible flow cell for serial synchrotron X-ray crystallography
- 2023
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Ingår i: Journal of Applied Crystallography. - : International Union of Crystallography (IUCr). - 1600-5767. ; 56, s. 449-460
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Tidskriftsartikel (refereegranskat)abstract
- Serial femtosecond crystallography was initially developed for roomtemperature X-ray diffraction studies of macromolecules at X-ray free electron lasers. When combined with tools that initiate biological reactions within microcrystals, time-resolved serial crystallography allows the study of structural changes that occur during an enzyme catalytic reaction. Serial synchrotron X-ray crystallography (SSX), which extends serial crystallography methods to synchrotron radiation sources, is expanding the scientific community using serial diffraction methods. This report presents a simple flow cell that can be used to deliver microcrystals across an X-ray beam during SSX studies. This device consists of an X-ray transparent glass capillary mounted on a goniometercompatible 3D-printed support and is connected to a syringe pump via lightweight tubing. This flow cell is easily mounted and aligned, and it is disposable so can be rapidly replaced when blocked. This system was demonstrated by collecting SSX data at MAX IV Laboratory from microcrystals of the integral membrane protein cytochrome c oxidase from Thermus thermophilus, from which an X-ray structure was determined to 2.12 angstrom resolution. This simple SSX platform may help to lower entry barriers for non-expert users of SSX.
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| 16. |
- Ghosh, Swagatha, et al.
(författare)
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A simple goniometer-compatible flow cell for serial synchrotron X-ray crystallography
- 2023
-
Ingår i: Journal of Applied Crystallography. - 0021-8898. ; 56:Pt 2, s. 449-460
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Tidskriftsartikel (refereegranskat)abstract
- Serial femtosecond crystallography was initially developed for room-temperature X-ray diffraction studies of macromolecules at X-ray free electron lasers. When combined with tools that initiate biological reactions within microcrystals, time-resolved serial crystallography allows the study of structural changes that occur during an enzyme catalytic reaction. Serial synchrotron X-ray crystallography (SSX), which extends serial crystallography methods to synchrotron radiation sources, is expanding the scientific community using serial diffraction methods. This report presents a simple flow cell that can be used to deliver microcrystals across an X-ray beam during SSX studies. This device consists of an X-ray transparent glass capillary mounted on a goniometer-compatible 3D-printed support and is connected to a syringe pump via lightweight tubing. This flow cell is easily mounted and aligned, and it is disposable so can be rapidly replaced when blocked. This system was demonstrated by collecting SSX data at MAX IV Laboratory from microcrystals of the integral membrane protein cytochrome c oxidase from Thermus thermophilus, from which an X-ray structure was determined to 2.12 Å resolution. This simple SSX platform may help to lower entry barriers for non-expert users of SSX.
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| 17. |
- Hakulinen, Jonna K, et al.
(författare)
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MraY-antibiotic complex reveals details of tunicamycin mode of action.
- 2017
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Ingår i: Nature chemical biology. - : Springer Science and Business Media LLC. - 1552-4469 .- 1552-4450. ; 13:3, s. 265-267
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Tidskriftsartikel (refereegranskat)abstract
- The rapid increase of antibiotic resistance has created an urgent need to develop novel antimicrobial agents. Here we describe the crystal structure of the promising bacterial target phospho-N-acetylmuramoyl-pentapeptide translocase (MraY) in complex with the nucleoside antibiotic tunicamycin. The structure not only reveals the mode of action of several related natural-product antibiotics but also gives an indication on the binding mode of the MraY UDP-MurNAc-pentapeptide and undecaprenyl-phosphate substrates.
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| 18. |
- Hammarin, Greger, 1981, et al.
(författare)
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No observable non-thermal effect of microwave radiation on the growth of microtubules
- 2024
-
Ingår i: SCIENTIFIC REPORTS. - 2045-2322. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Despite widespread public interest in the health impact of exposure to microwave radiation, studies of the influence of microwave radiation on biological samples are often inconclusive or contradictory. Here we examine the influence of microwave radiation of frequencies 3.5 GHz, 20 GHz and 29 GHz on the growth of microtubules, which are biological nanotubes that perform diverse functions in eukaryotic cells. Since microtubules are highly polar and can extend several micrometres in length, they are predicted to be sensitive to non-ionizing radiation. Moreover, it has been speculated that tubulin dimers within microtubules might rapidly toggle between different conformations, potentially participating in computational or other cooperative processes. Our data show that exposure to microwave radiation yields a microtubule growth curve that is distorted relative to control studies utilizing a homogeneous temperature jump. However, this apparent effect of non-ionizing radiation is reproduced by control experiments using an infrared laser or hot air to heat the sample and thereby mimic the thermal history of samples exposed to microwaves. As such, no non-thermal effects of microwave radiation on microtubule growth can be assigned. Our results highlight the need for appropriate control experiments in biophysical studies that may impact on the sphere of public interest.
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| 19. |
- Hao, Meng-Shu, et al.
(författare)
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Structural and biochemical analysis of family 92 carbohydrate-binding modules uncovers multivalent binding to β-glucans
- 2024
-
Ingår i: Nature Communications. - : Springer Nature. - 2041-1723 .- 2041-1723. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Carbohydrate-binding modules (CBMs) are non-catalytic proteins found appended to carbohydrate-active enzymes. Soil and marine bacteria secrete such enzymes to scavenge nutrition, and they often use CBMs to improve reaction rates and retention of released sugars. Here we present a structural and functional analysis of the recently established CBM family 92. All proteins analysed bind preferentially to β−1,6-glucans. This contrasts with the diversity of predicted substrates among the enzymes attached to CBM92 domains. We present crystal structures for two proteins, and confirm by mutagenesis that tryptophan residues permit ligand binding at three distinct functional binding sites on each protein. Multivalent CBM families are uncommon, so the establishment and structural characterisation of CBM92 enriches the classification database and will facilitate functional prediction in future projects. We propose that CBM92 proteins may cross-link polysaccharides in nature, and might have use in novel strategies for enzyme immobilisation.
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| 20. |
- Hering, Jenny, et al.
(författare)
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Exploring the Active Site of the Antibacterial Target MraY by Modified Tunicamycins.
- 2020
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Ingår i: ACS chemical biology. - : American Chemical Society (ACS). - 1554-8937 .- 1554-8929. ; 15:11, s. 2885-2895
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Tidskriftsartikel (refereegranskat)abstract
- The alarming growth of antibiotic resistance that is currently ongoing is a serious threat to human health. One of the most promising novel antibiotic targets is MraY (phospho-MurNAc-pentapeptide-transferase), an essential enzyme in bacterial cell wall synthesis. Through recent advances in biochemical research, there is now structural information available for MraY, and for its human homologue GPT (GlcNAc-1-P-transferase), that opens up exciting possibilities for structure-based drug design. The antibiotic compound tunicamycin is a natural product inhibitor of MraY that is also toxic to eukaryotes through its binding to GPT. In this work, we have used tunicamycin and modified versions of tunicamycin as tool compounds to explore the active site of MraY and to gain further insight into what determines inhibitor potency. We have investigated tunicamycin variants where the following motifs have been modified: the length and branching of the tunicamycin fatty acyl chain, the saturation of the fatty acyl chain, the 6″-hydroxyl group of the GlcNAc ring, and the ring structure of the uracil motif. The compounds are analyzed in terms of how potently they bind to MraY, inhibit the activity of the enzyme, and affect the protein thermal stability. Finally, we rationalize these results in the context of the protein structures of MraY and GPT.
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| 21. |
- Hering, Jenny, et al.
(författare)
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Structural basis for selective inhibition of antibacterial target MraY, a membrane-bound enzyme involved in peptidoglycan synthesis
- 2018
-
Ingår i: Drug Discovery Today. - : Elsevier BV. - 1359-6446 .- 1878-5832. ; 23:7, s. 1426-1435
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Forskningsöversikt (refereegranskat)abstract
- The rapid growth of antibiotic-resistant bacterial infections is of major concern for human health. Therefore, it is of great importance to characterize novel targets for the development of antibacterial drugs. One promising protein target is MraY (UDP-N-acetylmuramyl-pentapeptide: undecaprenyl phosphate N-acetylmuramyl-pentapeptide-1-phosphate transferase or MurNAc-1-P-transferase), which is essential for bacterial cell wall synthesis. Here, we summarize recent breakthroughs in structural studies of bacterial MraYs and the closely related human GPT (UDP-N-acetylglucosamine: dolichyl phosphate N-acetylglucosamine-1-phosphate transferase or GlcNAc-1-P-transferase). We present a detailed comparison of interaction modes with the natural product inhibitors tunicamycin and muraymycin D2. Finally, we speculate on possible routes to design an antibacterial agent in the form of a potent and selective inhibitor against MraY.
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| 22. |
- Kmezik, Cathleen, 1991, et al.
(författare)
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A polysaccharide utilization locus from the gut bacterium Dysgonomonas mossii encodes functionally distinct carbohydrate esterases.
- 2021
-
Ingår i: The Journal of biological chemistry. - : Elsevier BV. - 1083-351X .- 0021-9258. ; 296
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Tidskriftsartikel (refereegranskat)abstract
- The gut microbiota plays a central role in human health by enzymatically degrading dietary fiber and concomitantly excreting short chain fatty acids that are associated with manifold health benefits. The polysaccharide xylan is abundant in dietary fiber but non-carbohydrate decorations hinder efficient cleavage by glycoside hydrolases (GHs) and need to be addressed by carbohydrate esterases (CEs). Enzymes from carbohydrate esterase families 1 and 6 (CE1 & 6) perform key roles in xylan degradation by removing feruloyl and acetate decorations, yet little is known about these enzyme families especially with regards to their diversity in activity. Bacteroidetes bacteria are dominant members of the microbiota and often encode their carbohydrate-active enzymes in multi-gene polysaccharide utilization loci (PULs). Here we present the characterization of three CEs found in a PUL encoded by the gut Bacteroidete Dysgonomonas mossii. We demonstrate that the CEs are functionally distinct, with one highly efficient CE6 acetyl esterase and two CE1 enzymes with feruloyl esterase activities. One multidomain CE1 enzyme contains two CE1 domains: an N-terminal domain feruloyl esterase, and a C-terminal domain with minimal activity on model substrates. We present the structure of the C-terminal CE1 domain with the carbohydrate binding module that bridges the two CE1 domains, as well as a complex of the same protein fragment with methyl ferulate. The investment of D. mossii in producing multiple CEs suggests that improved accessibility of xylan for GHs as well as cleavage of covalent polysaccharide-polysaccharide and lignin-polysaccharide bonds are important enzyme activities in the gut environment.
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| 23. |
- Mazurkewich, Scott, 1982, et al.
(författare)
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Structural insights of the enzymes from the chitin utilization locus of Flavobacterium johnsoniae
- 2020
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10:1
-
Tidskriftsartikel (refereegranskat)abstract
- Chitin is one of the most abundant renewable organic materials found on earth. The chitin utilization locus in Flavobacterium johnsoniae, which encodes necessary proteins for complete enzymatic depolymerization of crystalline chitin, has recently been characterized but no detailed structural information on the enzymes was provided. Here we present protein structures of the F. johnsoniae chitobiase (FjGH20) and chitinase B (FjChiB). FjGH20 is a multi-domain enzyme with a helical domain not before observed in other chitobiases and a domain organization reminiscent of GH84 (beta -N-acetylglucosaminidase) family members. The structure of FjChiB reveals that the protein lacks loops and regions associated with exo-acting activity in other chitinases and instead has a more solvent accessible substrate binding cleft, which is consistent with its endo-chitinase activity. Additionally, small angle X-ray scattering data were collected for the internal 70 kDa region that connects the N- and C-terminal chitinase domains of the unique 158 kDa multi-domain chitinase A (FjChiA). The resulting model of the molecular envelope supports bioinformatic predictions of the region comprising six domains, each with similarities to either Fn3-like or Ig-like domains. Taken together, the results provide insights into chitin utilization by F. johnsoniae and reveal structural diversity in bacterial chitin metabolism.
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| 24. |
- Mazurkewich, Scott, 1982, et al.
(författare)
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Structure of a C1/C4-oxidizing AA9 lytic polysaccharide monooxygenase from the thermophilic fungus Malbranchea cinnamomea
- 2021
-
Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 77, s. 1019-1026
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Tidskriftsartikel (refereegranskat)abstract
- The thermophilic fungus Malbranchea cinnamomea contains a host of enzymes that enable its ability as an efficient degrader of plant biomass and that could be mined for industrial applications. This thermophilic fungus has been studied and found to encode eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), which collectively possess different substrate specificities for a range of plant cell-wall-related polysaccharides and oligosaccharides. To gain greater insight into the molecular determinants defining the different specificities, structural studies were pursued and the structure of McAA9F was determined. The enzyme contains the immunoglobulin-like fold typical of previously solved AA9 LPMO structures, but contains prominent differences in the loop regions found on the surface of the substrate-binding site. Most significantly, McAA9F has a broad substrate specificity, with activity on both crystalline and soluble polysaccharides. Moreover, it contains a small loop in a region where a large loop has been proposed to govern specificity towards oligosaccharides. The presence of the small loop leads to a considerably flatter and more open surface that is likely to enable the broad specificity of the enzyme. The enzyme contains a succinimide residue substitution, arising from intramolecular cyclization of Asp10, at a position where several homologous members contain an equivalent residue but cyclization has not previously been observed. This first structure of an AA9 LPMO from M. cinnamomea aids both the understanding of this family of enzymes and the exploration of the repertoire of industrially relevant lignocellulolytic enzymes from this fungus.
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| 25. |
- Mazurkewich, S., et al.
(författare)
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Structure of a C1/C4-oxidizing AA9 lytic polysaccharide monooxygenase from the thermophilic fungus Malbranchea cinnamomea
- 2021
-
Ingår i: Acta Crystallographica Section D-Structural Biology. - : International Union of Crystallography (IUCr). - 2059-7983. ; 77, s. 1019-1026
-
Tidskriftsartikel (refereegranskat)abstract
- The thermophilic fungus Malbranchea cinnamomea contains a host of enzymes that enable its ability as an efficient degrader of plant biomass and that could be mined for industrial applications. This thermophilic fungus has been studied and found to encode eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), which collectively possess different substrate specificities for a range of plant cell-wall-related polysaccharides and oligosaccharides. To gain greater insight into the molecular determinants defining the different specificities, structural studies were pursued and the structure of McAA9F was determined. The enzyme contains the immunoglobulin-like fold typical of previously solved AA9 LPMO structures, but contains prominent differences in the loop regions found on the surface of the substrate-binding site. Most significantly, McAA9F has a broad substrate specificity, with activity on both crystalline and soluble polysaccharides. Moreover, it contains a small loop in a region where a large loop has been proposed to govern specificity towards oligosaccharides. The presence of the small loop leads to a considerably flatter and more open surface that is likely to enable the broad specificity of the enzyme. The enzyme contains a succinimide residue substitution, arising from intramolecular cyclization of Asp10, at a position where several homologous members contain an equivalent residue but cyclization has not previously been observed. This first structure of an AA9 LPMO from M. cinnamomea aids both the understanding of this family of enzymes and the exploration of the repertoire of industrially relevant lignocellulolytic enzymes from this fungus.
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| 26. |
- Neutze, Richard, 1969, et al.
(författare)
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Membrane protein structural biology using X-ray free electron lasers
- 2015
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Ingår i: Current Opinion in Structural Biology. - : Elsevier BV. - 0959-440X .- 1879-033X. ; 33, s. 115-125
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Tidskriftsartikel (refereegranskat)abstract
- © 2015 . Membrane protein structural biology has benefitted tremendously from access to micro-focus crystallography at synchrotron radiation sources. X-ray free electron lasers (XFELs) are linear accelerator driven X-ray sources that deliver a jump in peak X-ray brilliance of nine orders of magnitude and represent a disruptive technology with potential to dramatically change the field. Membrane proteins were amongst the first macromolecules to be studied with XFEL radiation and include proof-of-principle demonstrations of serial femtosecond crystallography (SFX), the observation that XFEL data can deliver damage free crystallographic structures, initial experiments towards recording structural information from 2D arrays of membrane proteins, and time-resolved SFX, time-resolved wide angle X-ray scattering and time-resolved X-ray emission spectroscopy studies. Conversely, serial crystallography methods are now being applied using synchrotron radiation. We believe that a context dependent choice of synchrotron or XFEL radiation will accelerate progress towards novel insights in understanding membrane protein structure and dynamics.
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| 27. |
- Popp, David, et al.
(författare)
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Flow-aligned, single-shot fiber diffraction using a femtosecond X-ray free-electron laser
- 2017
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Ingår i: CYTOSKELETON. - : WILEY. - 1949-3584 .- 1949-3592. ; 74:12, s. 472-481
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Tidskriftsartikel (refereegranskat)abstract
- A major goal for X-ray free-electron laser (XFEL) based science is to elucidate structures of biological molecules without the need for crystals. Filament systems may provide some of the first single macromolecular structures elucidated by XFEL radiation, since they contain one-dimensional translational symmetry and thereby occupy the diffraction intensity region between the extremes of crystals and single molecules. Here, we demonstrate flow alignment of as few as 100 filaments (Escherichia coli pili, F-actin, and amyloid fibrils), which when intersected by femtosecond X-ray pulses result in diffraction patterns similar to those obtained from classical fiber diffraction studies. We also determine that F-actin can be flow-aligned to a disorientation of approximately 5 degrees. Using this XFEL-based technique, we determine that gelsolin amyloids are comprised of stacked -strands running perpendicular to the filament axis, and that a range of order from fibrillar to crystalline is discernable for individual -synuclein amyloids.
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| 28. |
- Price, Neil P J, et al.
(författare)
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Branched Chain Lipid Metabolism As a Determinant of the N-Acyl Variation of Streptomyces Natural Products.
- 2021
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Ingår i: ACS chemical biology. - : American Chemical Society (ACS). - 1554-8937 .- 1554-8929. ; 16:1, s. 116-124
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Tidskriftsartikel (refereegranskat)abstract
- Branched-chain fatty acids (BCFA) are encountered in Gram-positive bacteria, but less so in other organisms. The bacterial BCFA in membranes are typically saturated, with both odd- and even-numbered carbon chain lengths, and with methyl branches at either the ω-1 (iso) or ω-2 (anteiso) positions. The acylation with BCFA also contributes to the structural diversity of microbial natural products and potentially modulates biological activity. For the tunicamycin (TUN) family of natural products, the toxicity toward eukaryotes is highly dependent upon N-acylation with trans-2,3-unsaturated BCFA. The loss of the 2,3-unsaturation gives modified TUN with reduced eukaryotic toxicity but crucially with retention of the synergistic enhancement of the β-lactam group of antibiotics. Here, we infer from genomics, mass spectrometry, and deuterium labeling that the trans-2,3-unsaturated TUN variants and the saturated cellular lipids found in TUN-producing Streptomyces are derived from the same pool of BCFA metabolites. Moreover, non-natural primers of BCFA metabolism are selectively incorporated into the cellular lipids of TUN-producing Streptomyces and concomitantly produce structurally novel neo-branched TUN N-acyl variants.
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| 29. |
- Rehling, Daniel, 1985-
(författare)
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Structural Studies of Proteins involved in Nucleotide Metabolism : Studies of a ribonucleotide reductase from A. aeolicus and NUDT15
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is separated into two parts. The first part concerns ribonucleotide reductase from Aquifex aeolicus. A. aeolicus is a hyperthermophilic bacterium that thrives at extremely high temperatures of 80-95 °C. We present the X-ray crystal structures of both the R1 and R2 subunits of this protein, which represents the first structure of a class Ia ribonucleotide reductase from a hyperthermophile and the first structure of an R1 from the NrdAh phylogenetic subclass. Several novel features were seen in the R1 structure such as the simultaneous binding of two ATP molecules in the ATP-cone domain as well as a novel “β-hairpin hook” feature which spans the dimer interface of the R1 protein. The gene encoding the R2 protein contains a self-cleaving intein domain. We examined two constructs of this protein, one with the sequence of the intein removed at the DNA level and the wild-type construct. Both crystal structures were found to be identical, showing the efficient cleavage of the intein domain in the wild-type construct. The second part of this thesis concerns the NUDIX hydrolase NUDT15. The physiological function of NUDT15 is still unknown, however certain mutations in this gene are associated with thiopurine intolerance in patients. Thiopurines are chemotherapeutic drugs used in the treatment of diseases such as acute lymphoblastic leukemia, the most common type of childhood leukemia, and inflammatory bowel disease. Thiopurine drugs are converted by the cell to the active metabolite 6-thio-dGTP which can then act as a substrate for DNA polymerase. Incorporation of these anti-metabolites into DNA produces the desired cytotoxic effects. We show that NUDT15 breaks down the active metabolites of these drugs which leads to a lowered effective dose. The absence of a functioning NUDT15 protein in patients that have inactivating mutations in the gene coding for NUDT15 results in a drastically increased effective dose of these compounds. A normal dose of a thiopurine drug can lead to severe and possibly life-threatening complications in these patients. The role of NUDT15 in thiopurine metabolism is established by in vitro and cellular data as well as the X-ray crystal structure of NUDT15 in complex with 6-thio-GMP. Acyclovir and ganciclovir are two antiviral drugs whose mechanism of action is similar to that of thiopurines. These drugs are also metabolized to their tri-phosphorylated forms and are then preferentially incorporated into viral DNA. Here again, we use in vitro, cellular and structural data to show that NUDT15 breaks down the active metabolites of these drugs. Two separate and structurally distinct lines of potent inhibitors for NUDT15 were developed with support of crystallographic studies. We show that cells are sensitized to both thiopurine and antiviral treatments in the presence of these inhibitors. Binding of our inhibitors to NUDT15 provided substantial thermal stabilization. The stabilizing effect of inhibitor binding enabled us to solve structures of the four most clinically relevant NUDT15 variants, thus elucidating the structural basis for the thiopurine sensitivity phenotype.
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| 30. |
- Safari, Cecilia, 1989, et al.
(författare)
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Time-resolved serial crystallography to track the dynamics of carbon monoxide in the active site of cytochrome c oxidase
- 2023
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Ingår i: Science advances. - 2375-2548. ; 9:49
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Tidskriftsartikel (refereegranskat)abstract
- Cytochrome c oxidase (CcO) is part of the respiratory chain and contributes to the electrochemical membrane gradient in mitochondria as well as in many bacteria, as it uses the energy released in the reduction of oxygen to pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography to study the structural response of the active site upon flash photolysis of carbon monoxide (CO) from the reduced heme a3 of ba3-type CcO. In contrast with the aa3-type enzyme, our data show how CO is stabilized on CuB through interactions with a transiently ordered water molecule. These results offer a structural explanation for the extended lifetime of the CuB-CO complex in ba3-type CcO and, by extension, the extremely high oxygen affinity of the enzyme.
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| 31. |
- Salomonsson, Lina, et al.
(författare)
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Deuterium isotope effect of proton pumping in cytochrome c oxidase
- 2008
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1777:4, s. 343-350
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Tidskriftsartikel (refereegranskat)abstract
- In mitochondria and many aerobic bacteria cytochrome c oxidase is the terminal enzyme of the respiratory chain where it catalyses the reduction of oxygen to water. The free energy released in this process is used to translocate (pump) protons across the membrane such that each electron transfer to the catalytic site is accompanied by proton pumping. To investigate the mechanism of electron–proton coupling in cytochrome c oxidase we have studied the pH-dependence of the kinetic deuterium isotope effect of specific reaction steps associated with proton transfer in wild-type and structural variants of cytochrome c oxidases in which amino-acid residues in proton-transfer pathways have been modified. In addition, we have solved the structure of one of these mutant enzymes, where a key component of the proton-transfer machinery, Glu286, was modified to an Asp. The results indicate that the P3 → F3 transition rate is determined by a direct proton-transfer event to the catalytic site. In contrast, the rate of the F3 → O4 transition, which involves simultaneous electron transfer to the catalytic site and is characteristic of any transition during CytcO turnover, is determined by two events with similar rates and different kinetic isotope effects. These reaction steps involve transfer of protons, that are pumped, via a segment of the protein including Glu286 and Arg481.
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| 32. |
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| 33. |
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| 34. |
- Sandelin, Emil, 1994, et al.
(författare)
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Characterization and evaluation of photolabile (μ-peroxo)(μ-hydroxo)bis[bis(bipyridyl)cobalt caged oxygen compounds to facilitate time-resolved crystallographic studies of cytochrome c oxidase
- 2024
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Ingår i: PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES. - 1474-905X .- 1474-9092. ; 23, s. 839-851
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Tidskriftsartikel (refereegranskat)abstract
- Photolabile (mu-peroxo)(mu-hydroxo)bis[bis(bipyridyl)-cobalt-based caged oxygen compounds have been synthesized and characterized by optical absorbance spectroscopy, X-ray crystallography. and the quantum yield and redox stability were investigated. Furthermore, conditions were established where redox incompatibilities encountered between caged oxygen compounds and oxygen-dependant cytochrome c oxidase (CcO) could be circumvented. Herein, we demonstrate that millimolar concentrations of molecular oxygen can be released from a caged oxygen compound with spatio-temporal control upon laser excitation, triggering enzymatic turnover in cytochrome c oxidase. Spectroscopic evidence confirms the attainment of a homogeneous reaction initiation at concentrations and conditions relevant for further crystallography studies. This was demonstrated by the oxidizing microcrystals of reduced CcO by liberation of millimolar concentrations of molecular oxygen from a caged oxygen compound. We believe this will expand the scope of available techniques for the detailed investigation of oxygen-dependant enzymes with its native substrate and facilitate further time-resolved X-ray based studies such as wide/small angle X-ray scattering and serial femtosecond crystallography.
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| 35. |
- Sharma, Amit, et al.
(författare)
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A simple adaptation to a protein crystallography station to facilitate difference X-ray scattering studies
- 2019
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Ingår i: Journal of Applied Crystallography. - : International Union of Crystallography (IUCr). - 0021-8898 .- 1600-5767. ; 52, s. 378-386
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Tidskriftsartikel (refereegranskat)abstract
- The X-ray crystallography station I911-2 at MAXLab II (Lund, Sweden) has been adapted to enable difference small- and wide-angle X-ray scattering (SAXS/WAXS) data to be recorded. Modifications to the beamline included a customized flow cell, a motorized flow cell holder, a helium cone, a beam stop, a sample stage and a sample delivery system. This setup incorporated external devices such as infrared lasers, LEDs and reaction mixers to induce conformational changes in macromolecules. This platform was evaluated through proof-of-principle experiments capturing light-induced conformational changes in phytochromes. A difference WAXS signature of conformational changes in a plant aquaporin was also demonstrated using caged calcium.
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| 36. |
- Sharma, Amit, et al.
(författare)
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A simple adaptation to a protein crystallography station to facilitate difference X-ray scattering studies
- 2019
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Ingår i: Journal of Applied Crystallography. - 1600-5767. ; 52:2, s. 378-386
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Tidskriftsartikel (refereegranskat)abstract
- The X-ray crystallography station I911-2 at MAXLab II (Lund, Sweden) has been adapted to enable difference small- and wide-angle X-ray scattering (SAXS/WAXS) data to be recorded. Modifications to the beamline included a customized flow cell, a motorized flow cell holder, a helium cone, a beam stop, a sample stage and a sample delivery system. This setup incorporated external devices such as infrared lasers, LEDs and reaction mixers to induce conformational changes in macromolecules. This platform was evaluated through proof-of-principle experiments capturing light-induced conformational changes in phytochromes. A difference WAXS signature of conformational changes in a plant aquaporin was also demonstrated using caged calcium.
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| 37. |
- Shilova, Anastasiia, et al.
(författare)
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Current status and future opportunities for serial crystallography at MAX IV Laboratory
- 2020
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Ingår i: Journal of Synchrotron Radiation. - Chichester : Wiley-Blackwell. - 0909-0495 .- 1600-5775. ; 27, s. 1095-1102
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Tidskriftsartikel (refereegranskat)abstract
- Over the last decade, serial crystallography, a method to collect complete diffraction datasets from a large number of microcrystals delivered and exposed to an X-ray beam in random orientations at room temperature, has been successfully implemented at X-ray free-electron lasers and synchrotron radiation facility beamlines. This development relies on a growing variety of sample presentation methods, including different fixed target supports, injection methods using gas-dynamic virtual-nozzle injectors and high-viscosity extrusion injectors, and acoustic levitation of droplets, each with unique requirements. In comparison with X-ray free-electron lasers, increased beam time availability makes synchrotron facilities very attractive to perform serial synchrotron X-ray crystallography (SSX) experiments. Within this work, the possibilities to perform SSX at BioMAX, the first macromolecular crystallography beamline at studies from the SSX user program: an implementation of a high-viscosity extrusion injector to perform room temperature serial crystallography at BioMAX using two solid supports - silicon nitride membranes (Silson, UK) and XtalTool (Jena Bioscience, Germany). Future perspectives for the dedicated serial crystallography beamline MicroMAX at MAX IV Laboratory, which will provide parallel and intense micrometre-sized X-ray beams, are discussed.
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| 38. |
- Sörensen Ristinmaa, Amanda, 1992, et al.
(författare)
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Structural diversity and substrate preferences of three tannase enzymes encoded by the anaerobic bacterium Clostridium butyricum
- 2022
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 298:4
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Tidskriftsartikel (refereegranskat)abstract
- Tannins are secondary metabolites that are enriched in the bark, roots, and knots in trees and are known to hinder microbial attack. The biological degradation of water-soluble gallotannins, such as tannic acid, is initiated by tannase enzymes (EC 3.1.1.20), which are esterases able to liberate gallic acid from aromatic-sugar complexes. However, only few tannases have previously been studied in detail. Here, for the first time, we biochemically and structurally characterize three tannases from a single organism, the anaerobic bacterium Clostridium butyricum, which inhabits both soil and gut environments. The enzymes were named CbTan1-3, and we show that each one exhibits a unique substrate preference on a range of galloyl ester model substrates; CbTan1 and 3 demonstrated preference toward galloyl esters linked to glucose, while CbTan2 was more promiscuous. All enzymes were also active on oak bark extractives. Furthermore, we solved the crystal structure of CbTan2 and produced homology models for CbTan1 and 3. In each structure, the catalytic triad and gallatebinding regions in the core domain were found in very similar positions in the active site compared with other bacterial tannases, suggesting a similar mechanism of action among these enzymes, though large inserts in each enzyme showcase overall structural diversity. In conclusion, the varied structural features and substrate specificities of the C. butyricum tannases indicate that they have different biological roles and could further be used in development of new valorization strategies for renewable plant biomass.
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