Abstract
Direct optical activation of microbial rhodopsins in deep biological tissue suffers from ineffective light delivery because visible light is strongly scattered and absorbed. NIR light has deeper tissue penetration, but NIR-activation requires a transducer that converts NIR light into visible light in proximity to proteins of interest. Lanthanide-doped upconversion nanoparticles (UCNPs) are ideal transducer as they absorb near-infrared (NIR) light and emit visible light. Therefore, UCNP-assisted excitation of microbial rhodopsins with NIR light has been intensively studied by electrophysiology technique. While electrophysiology is a powerful method to test the functional performance of microbial rhodopsins, conformational changes associated with the NIR light illumination in the presence of UCNPs remain poorly understood. Since UCNPs have generally multiple emission peaks at different wavelengths, it is important to reveal if UCNP-generated visible light induces similar structural changes of microbial rhodopsins as conventional visible light illumination does. Here, we synthesize the lanthanide-doped UCNPs that convert NIR light to blue light. Using these NIR-to-blue UCNPs, we monitor the NIR-triggered conformational changes in sensory rhodopsin II from Natronomonas pharaonis (NpSRII), blue light-sensitive microbial rhodospsin, by FTIR spectroscopy. FTIR difference spectrum of NpSRII was recorded under two different excitation conditions: (ⅰ) with conventional blue light, (ⅱ) with UCNP-generated blue light upon NIR excitation. Both spectra display similar spectral features characteristic of the long-lived M photointermediate state during the photocycle of NpSRII. This study demonstrates that NIR-activation of NpSRII mediated by UCNPs takes place in a similar way to direct blue light activation of NpSRII.
Highlights
Microbial rhodopsins are retinal-containing membrane proteins pivotal in cellular behaviors
The synthesized lanthanide-doped upconversion nanoparticles (UCNPs) were characterized by transmission electron microscopy (TEM), powder X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDX), and FTIR spectroscopy
We have synthesized the lanthanide-doped UCNPs (NaYF4: 20 mol% Yb3+/0.5 mol% Tm3+) that absorb 980 nm NIR light and emit UV and blue light. With these UCNPs, we have conducted the FTIR spectroscopic measurements on the NIR activation of NpSRII to probe the conformational changes in the protein
Summary
Microbial rhodopsins are retinal-containing membrane proteins pivotal in cellular behaviors. Making good use of the sensitivity to visible light, microbial rhodopsins present powerful tools in optogenetics, a method that exploits light to control cellular responses. Protein Activation by Near-Infrared Light tissue (Yaroslavsky et al, 2002; Bashkatov et al, 2005; Knöpfel et al, 2010; Lin, 2011; Häusser, 2014), is challenging and ways to effectively deliver light to the target protein in deep tissue must be found. Engineered red light-active optogenetic proteins (Lin et al, 2013; Chuong et al, 2014; Klapoetke et al, 2014; Marshel et al, 2019) have gained deeper tissue penetration, but effective light delivery still relies on invasive approaches
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