Design of a light-gated proton channel based on the crystal structure of Coccomyxa rhodopsin

2019 ◽  
Vol 12 (573) ◽  
pp. eaav4203 ◽  
Author(s):  
Roman Fudim ◽  
Michal Szczepek ◽  
Johannes Vierock ◽  
Arend Vogt ◽  
Andrea Schmidt ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Host Cells ◽  
Proton Channel ◽  
Voltage Sensitivity ◽  
Heterologous Host ◽  
Time Resolved ◽  
Coccomyxa Subellipsoidea ◽  
Single Protein ◽  
Distinct Features ◽  
Channel Currents

The light-driven proton pump Coccomyxa subellipsoidea rhodopsin (CsR) provides—because of its high expression in heterologous host cells—an opportunity to study active proton transport under controlled electrochemical conditions. In this study, solving crystal structure of CsR at 2.0-Å resolution enabled us to identify distinct features of the membrane protein that determine ion transport directivity and voltage sensitivity. A specific hydrogen bond between the highly conserved Arg83 and the nearby nonconserved tyrosine (Tyr14) guided our structure-based transformation of CsR into an operational light-gated proton channel (CySeR) that could potentially be used in optogenetic assays. Time-resolved electrophysiological and spectroscopic measurements distinguished pump currents from channel currents in a single protein and emphasized the necessity of Arg83 mobility in CsR as a dynamic extracellular barrier to prevent passive conductance. Our findings reveal that molecular constraints that distinguish pump from channel currents are structurally more confined than was generally expected. This knowledge might enable the structure-based design of novel optogenetic tools, which derive from microbial pumps and are therefore ion specific.

scholarly journals Time-resolved transcriptional profiling of Trichinella-infected murine myocytes helps to elucidate host–pathogen interactions in the muscle stage

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Xiaoxiang Hu ◽  
Xiaolei Liu ◽  
Chen Li ◽  
Yulu Zhang ◽  
Chengyao Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Biology ◽  
Trichinella Spiralis ◽  
Expression Patterns ◽  
Muscle Cells ◽  
Host Cells ◽  
Initial Reaction ◽  
Global Changes ◽  
Mammalian Skeletal Muscle ◽  
Time Resolved

Abstract Background Parasites of the genus Trichinella are the pathogenic agents of trichinellosis, which is a widespread and severe foodborne parasitic disease. Trichinella spiralis resides primarily in mammalian skeletal muscle cells. After invading the cells of the host organism, T. spiralis must elude or invalidate the host’s innate and adaptive immune responses to survive. It is necessary to characterize the pathogenesis of trichinellosis to help to prevent the occurrence and further progression of this disease. The aims of this study were to elucidate the mechanisms of nurse cell formation, pathogenesis and immune evasion of T. spiralis, to provide valuable information for further research investigating the basic cell biology of Trichinella-infected muscle cells and the interaction between T. spiralis and its host. Methods We performed transcriptome profiling by RNA sequencing to identify global changes at 1, 3, 7, 10 and 15 days post-infection (dpi) in gene expression in the diaphragm after the parasite entered and persisted within the murine myocytes; the mice were infected by intravenous injection of newborn larvae. Gene expression analysis was based on the alignment results. Differentially expressed genes (DEGs) were identified based on their expression levels in various samples, and functional annotation and enrichment analysis were performed. Results The most extensive and dynamic gene expression responses in host diaphragms were observed during early infection (1 dpi). The number of DEGs and genes annotated in the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology databases decreased significantly in the infected mice compared to the uninfected mice at 3 and 7 dpi, suddenly increased sharply at 10 dpi, and then decreased to a lower level at 15 dpi, similar to that observed at 3 and 7 dpi. The massive initial reaction of the murine muscle cells to Trichinella infection steadied in the later stages of infection, with little additional changes detected for the remaining duration of the studied process. Although there were hundreds of DEGs at each time point, only 11 genes were consistently up- or downregulated at all 5 time points. Conclusions The gene expression patterns identified in this study can be employed to characterize the coordinated response of T. spiralis-infected myocytes in a time-resolved manner. This comprehensive dataset presents a distinct and sensitive picture of the interaction between host and parasite during intracellular infection, which can help to elucidate how pathogens evade host defenses and coordinate the biological functions of host cells to survive in the mammalian environment.

scholarly journals Time-Resolved Transcriptional Profiling of Epithelial Cells Infected by Intracellular Acinetobacter baumannii

2021 ◽  
Vol 9 (2) ◽  
pp. 354
Author(s):  
Nuria Crua Asensio ◽  
Javier Macho Rendón ◽  
Marc Torrent Burgas
Keyword(s):  
Acinetobacter Baumannii ◽  
Transcriptional Profiling ◽  
Multidrug Resistant ◽  
Host Cells ◽  
Resistant Bacteria ◽  
Multidrug Resistant Bacteria ◽  
Time Resolved ◽  
Antibiotic Resistant ◽  
Common Features ◽  
Profile Changes

The rise in the number of antibiotic-resistant bacteria has become a serious threat to health, making it important to identify, characterize and optimize new molecules to help us to overcome the infections they cause. It is well known that Acinetobacter baumannii has a significant capacity to evade the actions of antibacterial drugs, leading to its emergence as one of the bacteria responsible for hospital and community-acquired infections. Nonetheless, how this pathogen infects and survives inside the host cell is unclear. In this study, we analyze the time-resolved transcriptional profile changes observed in human epithelial HeLa cells after infection by A. baumannii, demonstrating how it survives in host cells and starts to replicate 4 h post infection. These findings were achieved by sequencing RNA to obtain a set of Differentially Expressed Genes (DEGs) to understand how bacteria alter the host cells’ environment for their own benefit. We also determine common features observed in this set of genes and identify the protein–protein networks that reveal highly-interacted proteins. The combination of these findings paves the way for the discovery of new antimicrobial candidates for the treatment of multidrug-resistant bacteria.

scholarly journals Time-Resolved X-ray Crystal Structure Analysis of Proteins.

1995 ◽  
Vol 35 (6) ◽  
pp. 245-248
Author(s):  
Shin-ichi ADACHI ◽  
Nobuhisa WATANABE
Keyword(s):  
Crystal Structure ◽  
Structure Analysis ◽  
Crystal Structure Analysis ◽  
Time Resolved ◽  
X Ray

Lateral deformations of a crystal of potassium acid phthalate in an external electric field

2021 ◽  
Vol 54 (5) ◽  
pp. 1317-1326
Author(s):  
Arsen Petrenko ◽  
Nataliya Novikova ◽  
Alexander Blagov ◽  
Anton Kulikov ◽  
Yury Pisarevskii ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Electrical Conductivity ◽  
Electric Field ◽  
External Electric Field ◽  
Applied Field ◽  
X Ray Diffraction ◽  
Time Resolved ◽  
X Ray ◽  
Potassium Acid Phthalate ◽  
Acid Phthalate

The anisotropy of deformations in potassium acid phthalate crystals arising under the action of an external electric field up to 1 kV mm−1 applied along the [001] polar axis was studied using X-ray diffraction methods at room temperature. Electrical conductivity was measured and rocking curves for reflections 400, 070 and 004 were obtained by time-resolved X-ray diffractometry in Laue and Bragg geometries. Two saturation processes were observed from the time dependences of the electrical conductivity. A shift in the diffraction peaks and a change in their intensity were found, which indicated a deformation of the crystal structure. Rapid piezoelectric deformation and reversible relaxation-like deformation, kinetically similar to the electrical conductivity of a crystal, were revealed. The deformation depended on the polarity and strength of the applied field. The deformation was more noticeable in the [100] direction and was practically absent in the [001] direction of the applied field. X-ray diffraction analysis revealed a disordered arrangement of potassium atoms, i.e. additional positions and vacancies. The heights of potential barriers between the positions of K+ ions and the paths of their possible migration in the crystal structure of potassium acid phthalate were determined. The data obtained by time-resolved X-ray diffractometry and X-ray structure analysis, along with additional electrophysical measurements, allow the conclusion that the migration of charge carriers (potassium cations) leads to lateral deformation of the crystal structure of potassium phthalate in an external electric field.

scholarly journals Export von Gefahrgut: Helicobacter pylori und sein CagA-Protein

BIOspektrum ◽  
2020 ◽  
Vol 26 (6) ◽  
pp. 597-599
Author(s):  
Clara Lettl ◽  
Wolfgang Fischer
Keyword(s):  
Helicobacter Pylori ◽  
Pathogenic Bacteria ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Bacterial Protein ◽  
Secretion Systems ◽  
Unusual Structure ◽  
Type Iv ◽  
Single Protein ◽  
Protein Transporters

Abstract Pathogenic bacteria often utilize type IV secretion systems to interact with host cells and to modify their microenvironment in a favourable way. The human pathogen Helicobacter pylori produces such a system to inject only a single protein, CagA, into gastric cells, but this injection represents a major risk factor for gastric cancer development. Here, we discuss the unusual structure of the Cag secretion nanomachine and other features that make it unique among bacterial protein transporters.

scholarly journals Crystal Structure of the Kinase Domain of MerTK in Complex with AZD7762 Provides Clues for Structure-Based Drug Development

2020 ◽  
Vol 21 (21) ◽  
pp. 7878
Author(s):  
Tae Hyun Park ◽  
Seung-Hyun Bae ◽  
Seoung Min Bong ◽  
Seong Eon Ryu ◽  
Hyonchol Jang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cancer Cell ◽  
Structural Information ◽  
Antiplatelet Agents ◽  
Binding Mode ◽  
Kinase Domain ◽  
Chemical Library ◽  
Time Resolved ◽  
Cancer Cell Death

Aberrant tyrosine-protein kinase Mer (MerTK) expression triggers prosurvival signaling and contributes to cell survival, invasive motility, and chemoresistance in many kinds of cancers. In addition, recent reports suggested that MerTK could be a primary target for abnormal platelet aggregation. Consequently, MerTK inhibitors may promote cancer cell death, sensitize cells to chemotherapy, and act as new antiplatelet agents. We screened an inhouse chemical library to discover novel small-molecule MerTK inhibitors, and identified AZD7762, which is known as a checkpoint-kinase (Chk) inhibitor. The inhibition of MerTK by AZD7762 was validated using an in vitro homogeneous time-resolved fluorescence (HTRF) assay and through monitoring the decrease in phosphorylated MerTK in two lung cancer cell lines. We also determined the crystal structure of the MerTK:AZD7762 complex and revealed the binding mode of AZD7762 to MerTK. Structural information from the MerTK:AZD7762 complex and its comparison with other MerTK:inhibitor structures gave us new insights for optimizing the development of inhibitors targeting MerTK.

scholarly journals Crystal structure of the type IV secretion system component CagX fromHelicobacter pylori

2017 ◽  
Vol 73 (3) ◽  
pp. 167-173 ◽  
Author(s):  
Jin Zhang ◽  
Fei Fan ◽  
Yanhe Zhao ◽  
Lifang Sun ◽  
Yadan Liu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Tertiary Structure ◽  
Protein Translocation ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Conjugative Plasmid ◽  
Dimensional Structure ◽  
System Component ◽  
Gastric Diseases ◽  
Type Iv

Helicobacter pylori, a Gram-negative bacterial pathogen prevalent in the human population, is the causative agent of severe gastric diseases. AnH. pyloritype IV secretion (T4S) system encoded by the cytotoxin-associated gene pathogenicity island (cagPAI) is responsible for communication with host cells. As a component of thecagPAI T4S system core complex, CagX plays an important role in virulence-protein translocation into the host cells. In this work, the crystal structure of the C-terminal domain of CagX (CagXct), which is a homologue of the VirB9 protein from the VirB/D4 T4S system, is presented. CagXct is only the second three-dimensional structure to be elucidated of a VirB9-like protein. Another homologue, TraO, which is encoded on theEscherichia coliconjugative plasmid pKM101, shares only 19% sequence identity with CagXct; however, there is a remarkable similarity in tertiary structure between these two β-sandwich protein domains. Most of the residues that are conserved between CagXct and TraO are located within the protein core and appear to be responsible for the preservation of this domain fold. The studies presented here will contribute to our understanding of different bacterial T4S systems.

scholarly journals Time-resolved in situ neutron diffraction studies of Li-ion battery materials

2014 ◽  
Vol 70 (a1) ◽  
pp. C353-C353 ◽  
Author(s):  
Neeraj Sharma
Keyword(s):  
Crystal Structure ◽  
Neutron Diffraction ◽  
Lithium Ion Batteries ◽  
Lithium Ion ◽  
Electrochemical Process ◽  
Atomic Scale ◽  
Structure Evolution ◽  
Time Resolved ◽  
In Situ Neutron Diffraction

Lithium-ion batteries are ubiquitous in society, used in everything from children's toys to mobile electronic devices, providing portable power solutions. There is a continuous drive for the improvement of these batteries to meet the demands of higher power devices and uses. A large proportion of the function of lithium-ion batteries arises from the electrodes, and these are in turn mediated by the atomic-scale perturbations or changes in the crystal structure during an electrochemical process (e.g. battery use). Therefore, a method to both understand battery function and propose ideas to improve their performance is to probe the electrode crystal structure evolution in situ while an electrochemical process is occurring inside a battery. Our work has utilized the benefits of in situ neutron diffraction (e.g. sensitivity towards lithium) to literally track the time-resolved evolution of lithium in electrode materials used in lithium-ion batteries (see Figure 1). With this knowledge we have been able to directly relate electrochemical properties such as capacity and differences in charge/discharge behaviour of a battery to the content and distribution of lithium in the electrode crystal structure. This talk will showcase some of our in situ investigations of materials in lithium-ion batteries, such as LiCoO2, LiFePO4, Li1+yMn2O4, LiNi0.5Mn1.5O4 and Li4Ti5O12/TiO2 electrodes. In addition, selected examples of our work using time-resolved in situ X-ray diffraction to probe other batteries types, such as primary lithium and secondary (rechargeable) sodium-ion batteries will be presented. Using time-resolved diffraction data, a comprehensive atomic-scale picture of battery functionality can be modelled and permutations can be made to the electrodes and electrochemical conditions to optimize battery performance. Therefore, crystallography and electrochemistry can mesh together to solve our energy needs.

scholarly journals A Single S4 Arginine is Sufficient for Voltage Sensitivity in the Hv1 Proton Channel

2010 ◽  
Vol 98 (3) ◽  
pp. 622a
Author(s):  
Ingrid Carvacho ◽  
I. Scott Ramsey ◽  
David E. Clapham
Keyword(s):  
Proton Channel ◽  
Voltage Sensitivity
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