CHAPTER 20: Photoactivated Structural Changes in Photoreceptor Membrane Proteins as Revealed by in situ Photoirradiation Solid-State NMR Spectroscopy

A novel in situ photoirradiation system for solid-state NMR measurements was developed and revealed photoactivated structural changes in photoreceptor membrane proteins. Using this method, we successfully identified the M-intermediates of pharaonis phoborhodopsin (ppR or sensory rhodopsin II) and the complex with the transducer (ppR/pHtrII) embedded in a model membrane. ¹³C NMR signals from [20-¹³C]retinal-ppR and ppR/pHtrII revealed that multiple M-intermediates (M₁, M₂ and M₃) with a 13-cis,15-anti retinal configuration coexist under continuous photoirradiation. One of the M-intermediates (M₃) was not an M-intermediate but probably an N-intermediate, as the M₃-intermediate was not activated by 365 nm LED light. The photocycle in [20-¹³C]retinal-SrSRI (sensory rhodopsin I) was examined using in situ photoirradiation solid-state NMR spectroscopy. The ¹³C NMR signal of the M intermediate was trapped at –40 °C by illumination with 520 (green) or 595 nm (orange) LED light, and the configuration of retinal was revealed to be 13-cis,15-anti. The M-intermediate was transformed into a P-intermediate by a second illumination with 365 nm (blue) light in a double-photon process. The P-intermediate was revealed to be the 13-cis,15-anti configuration of retinal. Further, the G-state could be transformed into the P-intermediate by illumination with 365 nm light through single- and/or double-photon processes.