Research Interests:
We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis... more
We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein…
Research Interests:
... the transient charge separation that occurs between the primary reactants P700+ and A(j. The resulting ... N 87 PsaL N 169 Eukaryotes only PsaG N 98 Prokaryotes onlt PsaMc C 30-32 PsaN ... genome but not in rice or tobacco genome; not... more
... the transient charge separation that occurs between the primary reactants P700+ and A(j. The resulting ... N 87 PsaL N 169 Eukaryotes only PsaG N 98 Prokaryotes onlt PsaMc C 30-32 PsaN ... genome but not in rice or tobacco genome; not found in any higher-plant PSI complexes ...
Research Interests:
To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray... more
To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 μm(3) in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 μm(3) in volume using the full...
Research Interests:
Research Interests:
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... more
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. Rat colon was exposed to tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) in Ussing chambers. Transepithelial resistance (R(t)) and (3)H-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). 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 x 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 x h(-1) x cm(-2). A new experimental IBD model with native colon in vitro is presented. One-day exposure to TNFalpha and IFNgamma 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.
Research Interests: Gastroenterology, Inflammatory Bowel Disease, In Vitro, Models, Intestinal Mucosa, and 15 morePermeability, Resistance, Animals, Intestinal barrier function, Male, Clinical Sciences, Rats, Rat, Tumor necrosis factor-alpha, Wistar Rats, Tight Junctions, Inflammatory Bowel Diseases, Interferon gamma, Claudins, and In Vitro Techniques
Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means... more
Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.
Research Interests:
Membrane proteins are extremely difficult to crystallize, however they are highly important proteins for cellular function. Photosystem I, one of the most complex membrane proteins solved to date took more than a decade to have a... more
Membrane proteins are extremely difficult to crystallize, however they are highly important proteins for cellular function. Photosystem I, one of the most complex membrane proteins solved to date took more than a decade to have a structure solved to molecular resolution. Large, well-ordered crystal growth is one of the major bottlenecks in structural determination by x-ray crystallography, due to the difficulty of making the "perfect" crystal. The development of femtosecond nanocrystallography, which uses a stream of fully hydrated nanocrystals to collect diffraction snapshots, effectively reduces this bottleneck[1] Photosystem II changed our biosphere via splitting water and evolving oxygen 2.5 billion years ago. Using femtosecond nanocrystallography we are developing a time-resolved femtosecond crystallography method [2] to unravel the mechanism of water splitting by determining the conformational changes that take place during the oxygen evolution process. Multiple crys...
Mikroreprod. eines Ms. Getr. Zählung : graph. Darst. Berlin, Techn. Univ., Diss., 1992.
G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal... more
G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-bi...
Research Interests:
Research Interests:
The potency of in vitro-added corticosteroids to stimulate electrogenic Na+ absorption (JNa, the Na+ absorptive short-circuit current blockable by 10(-4) M amiloride) was determined in rat late distal colon. JNa was determined 8 h after... more
The potency of in vitro-added corticosteroids to stimulate electrogenic Na+ absorption (JNa, the Na+ absorptive short-circuit current blockable by 10(-4) M amiloride) was determined in rat late distal colon. JNa was determined 8 h after steroid addition from the drop in short-circuit current caused by 10(-4) M amiloride. The concentration dependency of JNa was obtained for seven corticosteroids and compared with that established for aldosterone. Apparent mineralocorticoid potencies as determined from apparent Michaelis-Menten constant (Km) values were as follows: aldosterone 1. 2 nM > RU-28362 20 nM = deoxycorticosterone 20 nM > deoxycortisol 36 nM >/= dexamethasone 37 nM > corticosterone 170 nM > cortisol 210 nM. These steroids exhibited Vmax values of 9-13 micromol. h-1. cm-2 and similar concentration dependencies. Hill coefficients were between 1.6 and 2.1, suggesting cooperative effects between activated receptors. We conclude that corticosteroids exhibit graded m...
Research Interests:
CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein ofHuman immunodeficiency virus 1(HIV-1), and has previously been shown to induce... more
CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein ofHuman immunodeficiency virus 1(HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed inEscherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of t...
Serial femtosecond crystallography (SFX) is a new emerging method, where X-ray diffraction data are collected from a fully hydrated stream of nano- or microcrystals of biomolecules in their mother liquor using high-energy, X-ray... more
Serial femtosecond crystallography (SFX) is a new emerging method, where X-ray diffraction data are collected from a fully hydrated stream of nano- or microcrystals of biomolecules in their mother liquor using high-energy, X-ray free-electron lasers. The success of SFX experiments strongly depends on the ability to grow large amounts of well-ordered nano/microcrystals of homogeneous size distribution. While methods to grow large single crystals have been extensively explored in the past, method developments to grow nano/microcrystals in sufficient amounts for SFX experiments are still in their infancy. Here, we describe and compare three methods (batch, free interface diffusion (FID) and FID centrifugation) for growth of nano/microcrystals for time-resolved SFX experiments using the large membrane protein complex photosystem II as a model system.
Research Interests:
Research Interests:
Research Interests: Water, Catalysis, Photosynthesis, Cyanobacteria, Membrane Proteins, and 15 moreEnergy Conversion, X Rays, Solar Energy, Electron Transfer, Plants, Electron Transport, photosystem I, CHEMICAL SCIENCES, Protein Complex Detection, Oxygen, Biological Process, Molecular Conformation, Photosystem II, Membrane Protein, and Light Harvesting
ABSTRACT Photosystem I is a large membrane protein complex that catalyzes the first step of oxygenic photosynthesis. It can be regarded as a solar energy converter that captures the light from the sun through a large core-antenna system... more
ABSTRACT Photosystem I is a large membrane protein complex that catalyzes the first step of oxygenic photosynthesis. It can be regarded as a solar energy converter that captures the light from the sun through a large core-antenna system of chlorophylls and carotenoids and transfers the energy into the center of the complex, where the energy is used to catalyze the light-driven transmembrane electron transfer from plastocyanin to ferredoxin.
Research Interests:
ABSTRACT Photosynthesis is an efficient natural process that converts solar energy into chemical energy. The ancestors of today’s cyanobacteria were the first to use water as the electron source for this process, which resulted in the... more
ABSTRACT Photosynthesis is an efficient natural process that converts solar energy into chemical energy. The ancestors of today’s cyanobacteria were the first to use water as the electron source for this process, which resulted in the release of oxygen into the formerly reducing atmosphere of Earth. The actual conversion of light energy into chemical energy occurs in two large membrane protein complexes, Photosystems I and II, where the light-induced charge seperation across the photosynthetic membrane takes place. This chapter describes and compares the structure of both cyanobacterial Photosystems and discusses their function in respect to the mechanisms of light harvesting, electron transfer and water splitting. KeywordsX-ray structure analysis–Photosynthesis–Photosystem I–Photosystem II–Membrane proteins–Electron transfer–Light harvesting–Water Oxidation
Research Interests:
Research Interests:
ABSTRACT Photosystem I is a large membrane protein complex that catalyzes the first step of light reactions in photosynthesis. It can be regarded as a solar energy converter that captures the light from the sun through a large... more
ABSTRACT Photosystem I is a large membrane protein complex that catalyzes the first step of light reactions in photosynthesis. It can be regarded as a solar energy converter that captures the light from the sun through a large core-antenna system of chlorophylls and carotenoids. It then transfers the excitation energy into the center of the complex, where this electronic energy is used to catalyze the light-driven transmembrane electron transfer from plastocyanin to ferredoxin. Photosystem I of cyanobacterial origin consists of 12 protein subunits, to which 127 cofactors are non-covalently bound. This chapter describes the structure and function of cyanobacterial Photosystem I, as revealed from the X-ray structure at 2.5 Å resolution.
CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce... more
CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form o...
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests: Chemical Engineering, Biophysics, Science, Biophysical Chemistry, Multidisciplinary, and 11 moreMacromolecular X-Ray Crystallography, Biological Sciences, Humans, Lasers, G protein-coupled receptors, Physical sciences, CHEMICAL SCIENCES, Time Factors, Protein Conformation, Biochemistry and cell biology, and G Protein Coupled Receptors
Research Interests:
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... more
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. Rat colon was exposed to tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) in Ussing chambers. Transepithelial resistance (R(t)) and (3)H-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). 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 x 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 x h(-1) x cm(-2). A new experimental IBD model with native colon in vitro is presented. One-day exposure to TNFalpha and IFNgamma 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.
Research Interests: Gastroenterology, Inflammatory Bowel Disease, In Vitro, Models, Intestinal Mucosa, and 15 morePermeability, Resistance, Animals, Intestinal barrier function, Male, Clinical Sciences, Rats, Rat, Tumor necrosis factor-alpha, Wistar Rats, Tight Junctions, Inflammatory Bowel Diseases, Interferon gamma, Claudins, and In Vitro Techniques
ABSTRACT Two large membrane protein complexes, Photosystem I and II (PS I and PS II), perform the first step in the conversion of the light energy from the sun into chemical energy: the light-induced transmembrane charge separation. They... more
ABSTRACT Two large membrane protein complexes, Photosystem I and II (PS I and PS II), perform the first step in the conversion of the light energy from the sun into chemical energy: the light-induced transmembrane charge separation. They function in series; PS II provides the strong positive redox potential for water oxidation, while PS I generates a strong negative redox potential, which makes it able to reduce ferredoxin and deliver the electrons for the reduction of hydrogen in the form of NADPH. The structural comparison of PS I and PS II sheds light on the evolution of oxygenic photosynthesis. Both Photosystems show similarities in their core structure, indicating that they have been derived from a common ancestor. Striking differences in the arrangement and coordination of cofactors and in their protein environment, however, may contain the secret to the functional differences between the Photosystems. In this article, we address how the oxygen-evolving complex may have developed, and the main similarities and differences in the electron carriers and the organization of the antenna systems of these two complexes.
Research Interests:
Research Interests:
Research Interests:
Research Interests:
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... more
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…