CONFORMATION AND DYNAMICS OF [3-C-13]ALA-LABELED BACTERIORHODOPSIN AND BACTERIOOPSIN, INDUCED BY INTERACTION WITH RETINAL AND ITS ANALOGS, AS STUDIED BY C-13 NUCLEAR-MAGNETIC-RESONANCE

C-13 nuclear magnetic resonance (NMR) spectra of [3-C-13]Ala-labeled b acteriorhodopsin (bR), bacterioopsin (bO), and regenerated bR with ret inal or bO complex with retinal analogs were recorded in order to gain insights into how the conformation and dynamics of apoprotein (bO) va ry with or without retinal or its analogs. First, we assigned the C-13 NMR peak resonating at 16.3 ppm to Ala 53 of both bR and bO, which ap pears to contact the side chain of Lys 216 at the site of the Schiff b ase in the former, utilizing the C-13 NMR peaks of A53V and A53G prote ins in comparison with those of wild-type bR and bO. Characteristic sp ectral differences between the apoprotein and bR were observed upon re moval of the retinal: the changes of the peak intensities at 16.4, 15. 9, and 16.9 ppm are notable. We found that the loops (17.4 ppm) and tr ansmembrane alpha(II) helical region (15.9 ppm) acquired motional free dom with a correlation time of 10(-5) s when the retinal was removed, as detected by proton spin-lattice relaxation times in the rotating fr ame. A C-13 NMR spectrum very similar to that of native bR was recorde d when bR was regenerated by addition of retinal to bO. On the other h and, the addition of the retinal analogs retinol or beta-ionone, which are bound in the retinal binding site but are incapable of forming a Schiff base to the apoprotein, caused distinct spectral changes differ ent from those of bR, as manifested from the displacements of C-13 che mical shifts. These spectral changes must be ascribed to significant c onformational changes of apoprotein at various locations in the protei n, including the site of Ala 53 induced by modified interaction betwee n the apoprotein and chromophore.