DSB repair pathway choice is regulated by recruitment of 53BP1 through cell cycle-dependent regulation of Sp1

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Crystal Structure of the Light-Driven Chloride Pump Halorhodopsin from Natronomonas pharaonis

The light-driven chloride pump halorhodopsin from Natronomonas pharaonis (phR) crystallised into the monoclinic space group C2, with a phR trimer per the asymmetric unit. Diffraction data at 2.0-Å resolution showed that the carotenoid bacterioruberin binds to crevices between adjacent protein subunits in the trimeric assembly. Besides seven transmembrane helices (A to G) that characterise archaeal rhodopsins, the phR protomer possesses an amphipathic α-helix (A′) at the N-terminus. This helix, together with a long loop between helices B and C, forms a hydrophobic cap that covers the extracellular surface and prevents a rapid ion exchange between the active centre and the extracellular medium. The retinal bound to Lys256 in helix G takes on an all-trans configuration with the Schiff base being hydrogen-bonded to a water molecule. The Schiff base also interacts with Asp252 and a chloride ion, the latter being fixed by two polar groups (Thr126 and Ser130) in helix C. In the anion uptake pathway, four ionisable residues (Arg123, Glu234, Arg176 and His100) and seven water molecules are aligned to form a long hydrogen-bonding network. Conversely, the cytoplasmic half is filled mostly by hydrophobic residues, forming a large energetic barrier against the transport of anion. The height of this barrier would be lowered substantially if the cytoplasmic half functions as a proton/HCl antiporter. Interestingly, there is a long cavity extending from the main-chain carbonyl of Lys256 to Thr71 in helix B. This cavity, which is commonly seen in halobacterial light-driven proton pumps, is one possible pathway that is utilised for a water-mediated proton transfer from the cytoplasmic medium to the anion, which is relocated to the cytoplasmic channel during the photocycle.     

Mechanism divergence in microbial rhodopsins

A fundamental design principle of microbial rhodopsins is that they share the same basic light-induced conversion between two conformers. Alternate access of the Schiff base to the outside and to the cytoplasm in the outwardly open “E” conformer and cytoplasmically open “C” conformer, respectively, combined with appropriate timing of pKa changes controlling Schiff base proton release and uptake make the proton path through the pumps vectorial. Phototaxis receptors in prokaryotes, sensory rhodopsins I and II, have evolved new chemical processes not found in their proton pump ancestors, to alter the consequences of the conformational change or modify the change itself. Like proton pumps, sensory rhodopsin II undergoes a photoinduced E → C transition, with the C conformer a transient intermediate in the photocycle. In contrast, one light-sensor (sensory rhodopsin I bound to its transducer HtrI) exists in the dark as the C conformer and undergoes a light-induced C → E transition, with the E conformer a transient photocycle intermediate. Current results indicate that algal phototaxis receptors channelrhodopsins undergo redirected Schiff base proton transfers and a modified E → C transition which, contrary to the proton pumps and other sensory rhodopsins, is not accompanied by the closure of the external half-channel. The article will review our current understanding of how the shared basic structure and chemistry of microbial rhodopsins have been modified during evolution to create diverse molecular functions: light-driven ion transport and photosensory signaling by protein–protein interaction and light-gated ion channel activity.     

Heterochromatic repeat clustering imposes a physical barrier on homologous recombination to prevent chromosomal translocations

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Soluble expression and purification of a functional harpin protein in Escherichia coli

The use of harpins in practical agricultural applications may enhance plant growth and induce disease resistance. However, few investigations focused on the optimal expression and purification of harpin. In this work, harpin protein fused with a thioredoxin tag and a hexahistidine tag was expressed in Escherichia coli BL21 (DE3) cells as a soluble form under the induction of 0.5 mmol/L isopropyl β-D-1-thiogalactopyranoside. The purity of the recombinant harpin was greater than 90% after one-step nickel-nitrilotriacetic acid affinity chromatography. The yield of purified TRX-harpin protein reached 17.1 mg per 100 mL of cell culture. TRX-harpin is thermostable and could trigger the hypersensitive response effect in tobacco, with an efficient dose as low as 30 μg/mL. The root lengths of TRX-harpin treated tobacco and wheat plants were nearly 1.6-fold and 1.8-fold longer, respectively, than plants treated with the empty vector preparation. Thus, using a N-terminal TRX-tagged fusion is an economic way to produce bioactive harpin.