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Papers by Ingo Grotjohann
Nature methods, 2014
We describe a method to measure ultrafast protein structural changes using time-resolved wide-ang... 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…
Annual Review of Plant Physiology and Plant Molecular Biology, 1992
... the transient charge separation that occurs between the primary reactants P700+ and A(j. The ... 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 ...
Encyclopedia of Biophysics, 2013
P2X receptors for ATP comprise a family of ligand-gated cation channels that are distinct from th... more P2X receptors for ATP comprise a family of ligand-gated cation channels that are distinct from the cys-loop and glutamate channel families. They are expressed in one form or another by almost all cell types and play physiological roles ranging from pain sensation to blood clotting. This entry gives an overview of purinergic transmission, the basic properties of P2X receptors, and finally summarizes the main features and roles of molecularly defined receptor subtypes.
F1000 - Post-publication peer review of the biomedical literature, 2010
Proceedings of the National Academy of Sciences of the United States of America, Feb 28, 2017
To understand how molecules function in biological systems, new methods are required to obtain at... 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...
Http Dx Doi Org 10 1080 00365520903131973, Sep 1, 2009
In Inflammatory bowel disease (IBD), elevated cytokines are responsible for disturbed intestinal ... 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.
Nature communications, Jan 17, 2015
Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic ac... 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.
Acta Crystallographica Section A Foundations and Advances, 2014
Membrane proteins are extremely difficult to crystallize, however they are highly important prote... 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...
F1000 - Post-publication peer review of the biomedical literature, 2010
Mikroreprod. eines Ms. Getr. Zählung : graph. Darst. Berlin, Techn. Univ., Diss., 1992.
Nature, Jan 22, 2015
G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin bi... 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...
Annals of the New York Academy of Sciences, 2000
The American journal of physiology, 1999
The potency of in vitro-added corticosteroids to stimulate electrogenic Na+ absorption (JNa, the ... 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...
IUCrJ, 2014
CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proxima... 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...
Nature methods, 2014
We describe a method to measure ultrafast protein structural changes using time-resolved wide-ang... 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…
Annual Review of Plant Physiology and Plant Molecular Biology, 1992
... the transient charge separation that occurs between the primary reactants P700+ and A(j. The ... 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 ...
Encyclopedia of Biophysics, 2013
P2X receptors for ATP comprise a family of ligand-gated cation channels that are distinct from th... more P2X receptors for ATP comprise a family of ligand-gated cation channels that are distinct from the cys-loop and glutamate channel families. They are expressed in one form or another by almost all cell types and play physiological roles ranging from pain sensation to blood clotting. This entry gives an overview of purinergic transmission, the basic properties of P2X receptors, and finally summarizes the main features and roles of molecularly defined receptor subtypes.
F1000 - Post-publication peer review of the biomedical literature, 2010
Proceedings of the National Academy of Sciences of the United States of America, Feb 28, 2017
To understand how molecules function in biological systems, new methods are required to obtain at... 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...
Http Dx Doi Org 10 1080 00365520903131973, Sep 1, 2009
In Inflammatory bowel disease (IBD), elevated cytokines are responsible for disturbed intestinal ... 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.
Nature communications, Jan 17, 2015
Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic ac... 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.
Acta Crystallographica Section A Foundations and Advances, 2014
Membrane proteins are extremely difficult to crystallize, however they are highly important prote... 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...
F1000 - Post-publication peer review of the biomedical literature, 2010
Mikroreprod. eines Ms. Getr. Zählung : graph. Darst. Berlin, Techn. Univ., Diss., 1992.
Nature, Jan 22, 2015
G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin bi... 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...
Annals of the New York Academy of Sciences, 2000
The American journal of physiology, 1999
The potency of in vitro-added corticosteroids to stimulate electrogenic Na+ absorption (JNa, the ... 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...
IUCrJ, 2014
CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proxima... 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...