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.

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TheBiophysicalSociety of Japan GeneralIncorporatedAssociation gtudy to Lmderstand the ptopagatien oCthe deLreanye ofsignal -e mvegt]gated via Leerdinated switehmg whether dephesphoiv]ation ofCheY P by ChLZ dind its po]ar tocaTvation is required tor the intracLllular signa]ing [n cheZ dclLLecl Lcll the switching of t"o motors did not coordinatL In thL cell expressing loealvation less mutant of ChLZ Lhe Looidmated switchmg thas obHeTved These rcsults mdiLate dephogphorylation of CheY P is requirLd foi the signaT propagation whilc polar loLa]ization ofChcZ "ould not be essential HoueNer to ullderstand the role ot ChLZ m signal propagation we sheuTd imestigatL thL detail efthe Loordinsted switchmg tor example thc measurement ofdelay timc of CCW to CIJif and CW to CC W sxvltching mdividually WL "ant to discugs this at annuat mecung 9 fiO--t2 OO Q ft$/ ReDm Q mutants with the replacement of the N-terminal rcgion (about 30 residues) to investigate the origin of the differencc in the phetoreaction rate. Ppr-PYP-Rc-PYP chimera (N-termiiial of Ppr-PYP and the rest of Rc-PYP) was not refined yet. The spectral properties are generally detemnined by the C-terminal domain except for Hh-PYP-Rc-PYP. The spectrum of the chimeTa shows similar double maxima shape to that of Rc-PYP, but the major peak is similar to Hh-PYP. The both Rc-PYP-Hh-PYP and Ppr-PYP-Hh-PYP slowed down the thermal back reaction from illuminated state as compared with Hh-PYP. Hh-PYP-Rc-PYP has faster back reaction than Rc-PYP. On the other hand, Hh-PYP-Ppr-PYP and Rc-PYP-Ppr-PYP has similar reaction Tate te Ppr-PYP. These results indicate thatreactionrateislargelycontrolledbytheN-tenninalspecificinteractionwith the other part.

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