| 1. |
- Amasheh, Maren, et al.
(author)
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Regulation of mucosal structure and barrier function in rat colon exposed to tumor necrosis factor alpha and interferon gamma in vitro : A novel model for studying the pathomechanisms of inflammatory bowel disease cytokines
- 2009
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In: SCANDINAVIAN JOURNAL OF GASTROENTEROLOGY. - : Informa UK Limited. - 0036-5521 .- 1502-7708. ; 44:10, s. 1226-1235
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Journal article (peer-reviewed)abstract
- Objective. In Inflammatory bowel disease (IBD), elevated cytokines are responsible for disturbed intestinal transport and barrier function. The mechanisms of cytokine action have usually been studied in cell culture models only; therefore the aim of this study was to establish an in vitro model based on native intestine to analyze distinct cytokine effects on barrier function, mucosal structure, and inherent regulatory mechanisms. Material and methods. Rat colon was exposed to tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) in Ussing chambers. Transepithelial resistance (R-t) and H-3-mannitol fluxes were measured for characterization of the paracellular pathway. Transcellular transport was analyzed by horseradish peroxidase (HRP) flux measurements. Expression and distribution of tight junction proteins were characterized in immunoblots and by means of confocal laser-scanning microscopy (LSM). Results. Colonic viability could be preserved for 20 h in a specialized in vitro set-up. This was sufficient to alter mucosal architecture with crypt surface reduction. R-t was decreased (101 +/- 10 versus 189 +/- 10 Omega . cm(2)) with a parallel increase in mannitol permeability after cytokine exposure. Tight junction proteins claudin-1, -5, -7, and occludin decreased (45 +/- 10%, 16 +/- 7%, 42 +/- 8%, and 42 +/- 13% of controls, respectively), while claudin- 2 increased to 208 +/- 32%. Occludin and claudin- 1 translocated from the plasma membrane to the cytoplasm. HRP flux increased from 0.73 +/- 0.09 to 8.55 +/- 2.92 pmol . h(-1) . cm(-2). Conclusions. A new experimental IBD model with native colon in vitro is presented. One-day exposure to TNFa and IFNg alters mucosal morphology and impairs epithelial barrier function by up-regulation of the paracellular pore-former claudin-2 and down-regulation of the barrier-builders claudin-1, -5, and -7. These alterations resemble changes seen in IBD and thus underline their prominent role in IBD pathogenicity.
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| 2. |
- Aquila, Andrew, et al.
(author)
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Time-resolved protein nanocrystallography using an X-ray free-electron laser
- 2012
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In: Optics Express. - 1094-4087. ; 20:3, s. 2706-2716
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Journal article (peer-reviewed)abstract
- We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.
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| 3. |
- Chapman, Henry N, et al.
(author)
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Femtosecond X-ray protein nanocrystallography.
- 2011
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In: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 470:7332, s. 73-7
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Journal article (peer-reviewed)abstract
- X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200 nm to 2 μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.
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| 4. |
- Johansson, Linda C, 1983, et al.
(author)
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Structure of a photosynthetic reaction centre determined by serial femtosecond crystallography.
- 2013
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In: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 4
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Journal article (peer-reviewed)abstract
- Serial femtosecond crystallography is an X-ray free-electron-laser-based method with considerable potential to have an impact on challenging problems in structural biology. Here we present X-ray diffraction data recorded from microcrystals of the Blastochloris viridis photosynthetic reaction centre to 2.8 Å resolution and determine its serial femtosecond crystallography structure to 3.5 Å resolution. Although every microcrystal is exposed to a dose of 33 MGy, no signs of X-ray-induced radiation damage are visible in this integral membrane protein structure.
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| 5. |
- Kupitz, Christopher, et al.
(author)
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Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser
- 2014
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In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 513:7517, s. 261-265
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Journal article (peer-reviewed)abstract
- Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth's oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the 'dangler' Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.
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| 6. |
- Lee, Ho-Hsien, et al.
(author)
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Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1
- 2014
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In: IUCrJ. - 2052-2525. ; 1:5, s. 305-317
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Journal article (peer-reviewed)abstract
- CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) andthe membrane-proximal region of gp41 (MPR), the transmembrane envelopeprotein ofHuman immunodeficiency virus 1(HIV-1), and has previously beenshown to induce the production of anti-HIV-1 antibodies with antiviralfunctions. To further improve the design of this candidate vaccine, X-raycrystallography experiments were performed to obtain structural informationabout this fusion protein. Several variants of CTB-MPR were designed,constructed and recombinantly expressed inEscherichia coli. The first variantcontained a flexible GPGP linker between CTB and MPR, and yielded crystalsthat diffracted to a resolution of 2.3 A ̊, but only the CTB region was detectedin the electron-density map. A second variant, in which the CTB was directlyattached to MPR, was shown to destabilize pentamer formation. A thirdconstruct containing a polyalanine linker between CTB and MPR proved tostabilize the pentameric form of the protein during purification. The purificationprocedure was shown to produce a homogeneously pure and monodispersesample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered inthe third dimension. Nanocrystals obtained using the same precipitant showedpromising X-ray diffraction to 5 A ̊resolution in femtosecond nanocrystallo-graphy experiments at the Linac Coherent Light Source at the SLAC NationalAccelerator Laboratory. The results demonstrate the utility of femtosecondX-ray crystallography to enable structural analysis based on nano/microcrystalsof a protein for which no macroscopic crystals ordered in three dimensions havebeen observed before.
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| 7. |
- Liu, Wei, et al.
(author)
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Serial Femtosecond Crystallography of G Protein-Coupled Receptors
- 2013
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In: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 342:6165, s. 1521-1524
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Journal article (peer-reviewed)abstract
- X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. We used an x-ray free-electron laser (XFEL) with individual 50-femtosecond-duration x-ray pulses to minimize radiation damage and obtained a high-resolution room-temperature structure of a human serotonin receptor using sub-10-micrometer microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared with the structure solved by using traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room-temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment.
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| 8. |
- Redecke, Lars, et al.
(author)
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Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser.
- 2013
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In: Science (New York, N.Y.). - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 339:6116, s. 227-30
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Journal article (peer-reviewed)abstract
- The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the "diffraction-before-destruction" approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals.
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| 9. |
- Weierstall, Uwe, et al.
(author)
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Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography
- 2014
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 5, s. 3309-
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Journal article (peer-reviewed)abstract
- Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously renewed source of material for serial femtosecond crystallography. Data collected from sub-10-mu m-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor.
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