Arisu Shigeta

Solid-State Nuclear Magnetic Resonance Structural Study of the Retinal-Binding Pocket in Sodium Ion Pump Rhodopsin

The recently identified Krokinobacter rhodopsin 2 (KR2) functions as a light-driven sodium ion pump. The structure of the retinal-binding pocket of KR2 offers important insights into the mechanisms of KR2, which has motif of Asn112, Asp116, and Gln123 (NDQ) that is common among sodium ion pump rhodopsins but is unique among other microbial rhodopsins. Here we present solid-state nuclear magnetic resonance (NMR) characterization of retinal and functionally important residues in the vicinity of retinal in the ground state. We assigned chemical shifts of retinal C14 and C20 atoms, and Tyr218Cζ, Lys255Cε, and the protonated Schiff base of KR2 in lipid environments at acidic and neutral pH. 15N NMR signals of the protonated Schiff base showed a twist around the N-Cε bond under neutral conditions, compared with other microbial rhodopsins. These data indicated that the location of the counterion Asp116 is one helical pitch toward the cytoplasmic side. In acidic environments, the 15N Schiff base signal was shifted to a lower field, indicating that protonation of Asp116 induces reorientation during interactions between the Schiff base and Asp116. In addition, the Tyr218 residue in the vicinity of retinal formed a weak hydrogen bond with Asp251, a temporary Na+-binding site during the photocycle. These features may indicate unique mechanisms of sodium ion pumps.

Magnetically Alignable Bicelles with Unprecedented Stability Using Tunable Surfactants Derived from Cholic Acid.

Five novel surfactants were prepared by modifying the three hydroxy groups of sodium cholate with triethylene glycol chains endcapped with an amide (SC-C1 , SC-n C4 , and SC-n C5 ) or a carbamoyl group (SC-On C4 and SC-Ot C4 ). The phase behavior of aqueous mixtures of these surfactants with 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) was systematically studied by 31 P NMR spectroscopy. The surfactants endcapped with carbamate groups (SC-On C4 and SC-Ot C4 ) formed magnetically alignable bicelles over unprecedentedly wide ranges of conditions, in terms of temperature (from 21-23 to >90 °C), lipid/surfactant ratio (from 5 to 8), total lipid content (5-20 wt %), and lipid type [DMPC, 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC), or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC)]. In conjunction with appropriate phospholipids, the carbamate-endcapped surfactants afforded unique bicelles, characterized by exceptional thermal stabilities (from 0 to >90 °C), biomimetic lipid compositions (DMPC/POPC=25:75 to 50:50), and extremely large 2 H quadrupole splittings (up to 71 Hz).