Rhodopsin-bestrophin fusion proteins from unicellular algae form gigantic pentameric ion channels.

Many organisms sense light using rhodopsins, photoreceptive proteins containing a retinal chromophore. Here we report the discovery, structure and biophysical characterization of bestrhodopsins, a microbial rhodopsin subfamily from marine unicellular algae, in which one rhodopsin domain of eight transmembrane helices or, more often, two such domains in tandem, are C-terminally fused to a bestrophin channel. Cryo-EM analysis of a rhodopsin-rhodopsin-bestrophin fusion revealed that it forms a pentameric megacomplex (~700 kDa) with five rhodopsin pseudodimers surrounding the channel in the center. Bestrhodopsins are metastable and undergo photoconversion between red- and green-absorbing or green- and UVA-absorbing forms in the different variants. The retinal chromophore, in a unique binding pocket, photoisomerizes from all-trans to 11-cis form. Heterologously expressed bestrhodopsin behaves as a light-modulated anion channel.

Gate-keeper of ion transport-a highly conserved helix-3 tryptophan in a channelrhodopsin chimera, C1C2/ChRWR.

Microbial rhodopsin is a large family of membrane proteins having seven transmembrane helices (TM1-7) with an all-trans retinal (ATR) chromophore that is covalently bound to Lys in the TM7. The Trp residue in the middle of TM3, which is homologous to W86 of bacteriorhodopsin (BR), is highly conserved among microbial rhodopsins with various light-driven functions. However, the significance of this Trp for the ion transport function of microbial rhodopsins has long remained unknown. Here, we replaced the W163 (BR W86 counterpart) of a channelrhodopsin (ChR), C1C2/ChRWR, which is a chimera between ChR1 and 2, with a smaller aromatic residue, Phe to verify its role in the ion transport. Under whole-cell patch clamp recordings from the ND7/23 cells that were transfected with the DNA plasmid coding human codon optimized C1C2/ChRWR (hWR) or its W163F mutant (hWR-W163F), the photocurrents were evoked by a pulsatile light at 475 nm. The ion-transporting activity of hWR was strongly altered by the W163F mutation in 3 points: (1) the H+ leak at positive membrane potential (V m) and its light-adaptation, (2) the attenuation of cation channel activity and (3) the manifestation of outward H+ pump activity. All of these results strongly suggest that W163 has a role in stabilizing the structure involved in the gating-on and -off of the cation channel, the role of "gate keeper". We can attribute the attenuation of cation channel activity to the incomplete gating-on and the H+ leak to the incomplete gating-off.

Transcription of Chirality from Metal-Organic Framework to Polythiophene.

Here, we report an unprecedented chirality transfer from a metal-organic framework (MOF) to a polymer. In this work, unsubstituted polythiophene (PTh) was prepared in the nanochannels of a chiral MOF. Circular dichroism spectroscopy revealed that nanoconfinement of the polymer chains could endow optically inactive PTh with a chiral nature. The thickness of polymer chain assemblies could be controlled by tuning the loading amount of PTh, which resulted in a drastic change in the chiroptical properties. Note that PTh liberated from the host still exhibited chirality even without the chiral support. Remarkably, the recovered PTh presented high thermal stability of chirality up to 250 °C. Our findings show that the encapsulation of the polymer chains in chiral MOFs is a simple and effective methodology not only to express the chirality of polymers but also to elucidate the inter- and intrachain chirality in polymer assemblies.