Akira Naito

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.

Unusual Dynamics of Alanine Residues in Polyalanine Regions with Staggered Packing Structure of Samia Cynthia Ricini Silk Fiber in Dry and Hydrated States Studied by 13C Solid-State NMR and Molecular Dynamics Simulation

Recently, the wild silkworm and spider dragline silks have been paid considerable attention as potentially valuable biomedical materials. Samia cynthia ricini is one of the wild silkworms and the primary structure of the silk fibroin (SF) consists of tandemly repeated polyalanine (poly-A:(A)12,13). Here, we report the unusual dynamical character observed in Ala Cβ groups in the poly-A region which forms an antiparallel-β-sheet structure with a staggered packing arrangement. The 13C spin-lattice relaxation ( T1s) and spin-spin relaxation times ( T2s) of Ala Cβ peaks in S. c. ricini SF fibers were observed in dry and hydrated states. The lowest field peak in Ala Cβ of the poly-A region showed 2 times longer T1 value and shorter correlation time than the other Ala Cβ peaks of the staggered packing structure, suggesting unusually fast hopping in methyl groups. Molecular dynamics simulations indicated that two of the Ala Cβ carbons out of eight existing in the unit cell of the staggered packing structure exhibited the fastest hopping motion in spite of the shortest Cβ-Cβ distance, indicating a geared hopping motion. T2 values of the hydrated and dry Ala Cβ peaks showed a similar value, indicating that the backbone motion of S. c. ricini SF fiber is not significantly affected by hydration.

Structural Analyses of Alanine Trimer and Tetramer Crystals with Antiparallel and Parallel β-Sheet Structures Using Solid-State 1H Spin-Diffusion 2D Correlation NMR Spectroscopy

Poly-l-alanine (PLA) sequences are key elements of the crystalline domains of spider dragline and wild silkworm silks. In the present work, 1H spin-diffusion two-dimensional (2D) correlation NMR spectra were observed for selectively deuterated (Ala)3 and (Ala)4 crystals to develop the analytical method for the structure of PLA sequences. The build-up curves of the cross peaks for three kinds of 1H pairs in selectively deuterated (Ala)3 and (Ala)4 crystals were observed to obtain spin-diffusion rate constant k j, k from relaxation master equations P i, j(τm). The k j, k values subsequently lead to effective interproton distance r j, keff (obs) values for individual proton-proton pairs, which include intra- and intermolecular contributions. The r j, keff (obs) values were compared to r j, keff (calc) values obtained from the experimentally determined atomic coordinates of antiparallel (AP) β-sheet (Ala)3 and (Ala)4 and parallel (P) β-sheet of (Ala)3 and (Ala)4 crystals. The agreement between the r j, keff (obs) and r j, keff (calc) values was good for AP β-sheet (Ala)3 and (Ala)4 crystals but poor for P β-sheet (Ala)3 and (Ala)4 crystals. These deviations were obtained from the interproton distances of the interchain contributions due to different packing arrangements. The packing arrangements of the PLA region are important when considering the relevant structure and the mechanical properties of silks.