Cytoplasmic surface structures of bacteriorhodopsin modified by site-directed mutations and cation binding as revealed by 13C NMR

Participation of the Surface Structure of Pharaonis Phoborhodopsin, ppR and its A149S and A149V mutants, Consisting of the C-terminal α-helix and E-F Loop, in the Complex-formation with the Cognate Transducer pHtrII, as Revealed by Site-directed 13C Solid

“…In contrast, it is also interesting to note that a single‐mutation in the E–F loop results in significantly modified surface and dynamics structures in bR which also include the C‐terminal α‐helix and the E–F loop, as manifested from the 13 C NMR spectra of A160G and A160V of bR mutants (corresponding to Ala149 in p pR) (Table 1) (25,29), by taking into account the well‐known structural homology between p pR and bR. It is, therefore, suggested that dynamics of the membrane surface residues including the E–F loop in the cytoplasmic side in p pR are strongly correlated with the manner of mutual interaction with p HtrII and its resulting biological function in relation to signal relay.…”

“…As a result, the organization of the cytoplasmic surface structures, including the C‐terminal α‐helix and the E–F loop as a function of metal ions, pH, temperature, etc . plays an essential role for the stabilization of the three‐dimensional structure of bR at ambient temperature (26–29). It is demonstrated in this study that the 13 C NMR signals of the C‐terminal α‐helical region were also recently identified for [3‐ 13 C]Ala‐ and [1‐ 13 C]Val‐labeled p pR and D75N mutant in the presence or absence of p HtrII(1–159) in egg phosphatidylcholine (PC) bilayers (see Fig.…”

Dynamics Change of Phoborhodopsin and Transducer by Activation: Study Using D75N Mutant of the Receptor by Site‐directed Solid‐state 13C NMR†

“…The former peak at 15.8 ppm was unequivocally assigned to Ala221 of the stem region of the C‐terminal α‐helix with reference to the conformation‐dependent displacement of peaks and also the peak‐position of the C‐terminal α‐helix portion of bR taking similar environment (Ala228 and 233) (40–42). The remaining 13 C NMR peaks at 16.7–16.9 ppm (lower field gray peaks) could be then ascribed to those of Ala228, 234, 236 and 238 in the C‐terminal α‐helical tip region, exhibiting the dynamics‐dependent 13 C shift because of low‐frequency fluctuation motion.…”

Surface and Dynamic Structures of Bacteriorhodopsin in a 2D Crystal, a Distorted or Disrupted Lattice, as Revealed by Site-directed Solid-state 13C NMR†

“…The surface structures are naturally altered either by site‐directed mutations at the C‐terminal α‐helix (R227Q) or at the loop (A160G, E166G and A168G), and by the manner of altered cation binding, as viewed from the 13 C chemical shifts of Ala 228 and 233 (C‐terminal α‐helix), Ala 103 (C–D loop) and Ala 160 (E–F loop). In contrast, the cytoplasmic ends of the B and F helices are found to undergo fluctuation motions of the order of 10 −5 s when such a surface structure is disrupted (54). To make proton uptake efficient during the photocycles, the following surface structure is proposed: the C‐terminal α‐helix of the wild‐type at ambient temperature is tilted toward the direction of the B and F helices.…”

“…This facilitates efficient proton uptake by preventing unnecessary fluctuations of the helices. Such a surface structure, however, is destabilized in that the C‐terminal α‐helix is straightforwardly extended from the helix G at low temperatures or in an M‐like state (54). This view is consistent with the previously published data for the “proton binding cluster” consisting of Asp 104, Glu 166 and Glu 224 (55–57).…”