Photoreaction Pathways of Bacteriorhodopsin and Its D96N Mutant as Revealed by in Situ Photoirradiation Solid-State NMR.

Bacteriorhodopsin (BR) functions as a light-driven proton pump that transitions between different states during the photocycle, such as all-trans (AT; BR568) and 13-cis, 15-syn (CS; BR548) state and K, L, M1, M2, N, and O intermediates. In this study, we used in situ photoirradiation 13C solid-state NMR to observe a variety of photo-intermediates and photoreaction pathways in [20-13C]retinal-WT-BR and its mutant [20-13C, 14-13C]retinal-D96N-BR. In WT-BR, the CS state converted to the CS* intermediate under photoirradiation with green light at -20 °C and consequently converted to the AT state in the dark. The AT state converted to the N intermediate under irradiation with green light. In D96N-BR, the CS state was converted to the CS* intermediate at -30 °C and consequently converted to the AT state. Simultaneously, the AT state converted to the M and L intermediates under green light illumination at -30 °C and subsequently converted to the AT state in the dark. The M intermediate was directly excited to the AT state by UV light illumination. We demonstrated that short-lived photo-intermediates could be observed in a stationary state using in situ photoirradiation solid-state NMR spectroscopy for WT-BR and D96N-BR, enabling insight into the light-driven proton pump activity of BR.

Dual-Color Fluorescence Switch-On Probe for Imaging G-Quadruplex and Double-Stranded DNA in Living Cells.

A tripodal quinone-cyanine dye having one donor and three acceptors, that is, one quinone and three N-methylbenzothiazolium moieties, QCy(MeBT)3, was synthesized by simple Knoevenagel condensation between 2-hydroxybenzene-1,3,5-tricarbaldehyde and N-methyl-2-methylbenzothiazolium iodide. The 700 nm (λex, 570 nm) and 600 nm (λex, 470 nm) fluorescence emission of QCy(MeBT)3 was significantly and individually enhanced with the addition of G-quadruplex (G4) DNA and double-stranded DNA (dsDNA), respectively. The results of docking simulations and the response against the viscosity change revealed that the dual-fluorescence response was caused by the difference in the binding mode of QCy(MeBT)3 depending on the DNA structure. The results of fluorescence microscopy imaging experiments using QCy(MeBT)3 suggested that G4 DNAs and dsDNAs in the cell nucleus can be imaged with near-infrared (NIR, 700 nm) and red (600 nm) fluorescence emissions. Furthermore, pyridostatin-induced G4 formation in the living cells can be imaged with NIR fluorescence. The results indicated that QCy(MeBT)3 has huge potential to be a NIR-fluorescent molecular probe for analyzing the structural dynamics of nucleic acids in living cells with a normal fluorescence microscope.