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Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition of abnormal heart rhythm (arrhythmia), induced by physical activity or stress. Mutations in ryanodine receptor 2 (RyR2), a Ca2+ release channel located in the sarcoplasmic reticulum (SR), or calsequestrin 2 (CASQ2), a SR Ca2+ binding protein, are linked to CPVT. For specific drug development and to study distinct arrhythmias, simple models are required to implement and analyze such mutations. Here, we introduced CPVT inducing mutations into the pharynx of Caenorhabditis elegans, which we previously established as an optogenetically paced heart model. By electrophysiology and video-microscopy, we characterized mutations in csq-1 (CASQ2 homologue) and unc-68 (RyR2 homologue). csq-1 deletion impaired pharynx function and caused missed pumps during 3.7 Hz pacing. Deletion mutants of unc-68, and in particular the point mutant UNC-68(R4743C), analogous to the established human CPVT mutant RyR2(R4497C), were unable to follow 3.7 Hz pacing, with progressive defects during long stimulus trains. The pharynx either locked in pumping at half the pacing frequency or stopped pumping altogether, possibly due to UNC-68 leakiness and/or malfunctional SR Ca2+ homeostasis. Last, we could reverse this ‘worm arrhythmia’ by the benzothiazepine S107, establishing the nematode pharynx for studying specific CPVT mutations and for drug screening.
We have determined the crystal structures of two decachlorocyclopentasilanes, namely bis(tetra-n-butylammonium) dichloride decachlorocyclopentasilane dichloromethane disolvate, 2C16H36N+·2Cl−·Si5Cl10·2CH2Cl2, (I), and bis(tetraethylammonium) dichloride decachlorocyclopentasilane dichloromethane disolvate, 2C8H20N+·2Cl−·Si5Cl10·2CH2Cl2, (II), both of which crystallize with discrete cations, anions, and solvent molecules. In (I), the complete decachlorocyclopentasilane ring is generated by a crystallographic twofold rotation axis. In (II), one cation is located on a general position and the other two are disordered about centres of inversion. These are the first structures featuring the structural motif of a five-membered cyclopentasilane ring coordinated from both sides by a chloride ion. The extended structures of (I) and (II) feature numerous C—H⋯Cl interactions. In (II), the N atoms are located on centres of inversion and as a result, the ethylene chains are disordered over equally occupied orientations.
Ion channel gating is essential for cellular homeostasis and is tightly controlled. In some eukaryotic and most bacterial ligand-gated K+ channels, RCK domains regulate ion fluxes. Until now, a single regulatory mechanism has been proposed for all RCK-regulated channels, involving signal transduction from the RCK domain to the gating area. Here, we present an inactive ADP-bound structure of KtrAB from Vibrio alginolyticus, determined by cryo-electron microscopy, which, combined with EPR spectroscopy and molecular dynamics simulations, uncovers a novel regulatory mechanism for ligand-induced action at a distance. Exchange of activating ATP to inactivating ADP triggers short helical segments in the K+-translocating KtrB dimer to organize into two long helices that penetrate deeply into the regulatory RCK domains, thus connecting nucleotide-binding sites and ion gates. As KtrAB and its homolog TrkAH have been implicated as bacterial pathogenicity factors, the discovery of this functionally relevant inactive conformation may advance structure-guided drug development.
The soluble loop BC region guides, but not dictates, the assembly of the transmembrane cytochrome b6
(2017)
Studying folding and assembly of naturally occurring α-helical transmembrane proteins can inspire the design of membrane proteins with defined functions. Thus far, most studies have focused on the role of membrane-integrated protein regions. However, to fully understand folding pathways and stabilization of α–helical membrane proteins, it is vital to also include the role of soluble loops. We have analyzed the impact of interhelical loops on folding, assembly and stability of the heme-containing four-helix bundle transmembrane protein cytochrome b6 that is involved in charge transfer across biomembranes. Cytochrome b6 consists of two transmembrane helical hairpins that sandwich two heme molecules. Our analyses strongly suggest that the loop connecting the helical hairpins is not crucial for positioning the two protein “halves” for proper folding and assembly of the holo-protein. Furthermore, proteolytic removal of any of the remaining two loops, which connect the two transmembrane helices of a hairpin structure, appears to also not crucially effect folding and assembly. Overall, the transmembrane four-helix bundle appears to be mainly stabilized via interhelical interactions in the transmembrane regions, while the soluble loop regions guide assembly and stabilize the holo-protein. The results of this study might steer future strategies aiming at designing heme-binding four-helix bundle structures, involved in transmembrane charge transfer reactions.
Cells respond to protein misfolding and aggregation in the cytosol by adjusting gene transcription and a number of post-transcriptional processes. In parallel to functional reactions, cellular structure changes as well; however, the mechanisms underlying the early adaptation of cellular compartments to cytosolic protein misfolding are less clear. Here we show that the mammalian ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP), if freed from chaperones during acute stress, can dock on cellular membranes thus performing a proteostasis sensor function. We reconstituted this process in vitro and found that mainly phosphatidic acid and phosphatidylinositol-4-phosphate enhance association of chaperone-free CHIP with liposomes. HSP70 and membranes compete for mutually exclusive binding to the tetratricopeptide repeat domain of CHIP. At new cellular locations, access to compartment-specific substrates would enable CHIP to participate in the reorganization of the respective organelles, as exemplified by the fragmentation of the Golgi apparatus (effector function).
The widespread application of human stem-cell-derived neurons for functional studies is impeded by complicated differentiation protocols, immaturity, and deficient optogene expression as stem cells frequently lose transgene expression over time. Here we report a simple but precise Cre-loxP-based strategy for generating conditional, and thereby stable, optogenetic human stem-cell lines. These cells can be easily and efficiently differentiated into functional neurons, and optogene expression can be triggered by administering Cre protein to the cultures. This conditional expression system may be applied to stem-cell-derived neurons whenever timed transgene expression could help to overcome silencing at the stem-cell level.
N-Allyltetramethylpiperidine is readily isomerized to the corresponding enamine by treatment with catalytic amounts of B(C6F5)3. It adds HB(C6F5)2 at the nucleophilic enamine carbon atom to form a C/B Lewis adduct. This reacts with two molar equivalents of carbon monoxide by selective head to tail coupling to give a five-membered C2O2B heterocycle. In contrast the enamine/HB(C6F5)2 Lewis pair reacts with two molar equiv. of nitric oxide by head to head coupling. This reaction probably proceeds via equilibrium with the corresponding vicinal N/B Lewis pair. Most products were characterized by X-ray diffraction.
Lieblingsbild
(2017)
Lieblingsbild
(2017)
Das Bild zeigt die Kryo-EM-Elektronendichtekarte des bakteriellen Kalium-Aufnahmesystems KtrAB mit ADP gebunden mit einer Auflösung von 6.6 Å. Das Bild ist mein Lieblingsbild, weil man bereits auf den ersten Blick eine dramatische Konformationsänderung im Vergleich zu einer früheren ATPgebundenen Struktur erkennen konnte, nämlich die Ausbildung langgestreckter α-Helices (hier gelb markiert), die die regulatorischen A-Untereinheiten (blau) mit den Kaliumionen-translozierenden B-Untereinheiten (grau) verbinden. ...