Probing intramolecular orientations in rhodopsin and metarhodopsin II by polarized infrared difference spectroscopy.
The light-induced conformational changes of rhodopsin, which lead to the formation of the G-protein activating metarhodopsin II intermediate, are studied by polarized attenuated total reflectance infrared difference spectroscopy. Orientations of protein groups as well as the retinylidene chromophore were calculated from the linear dichroism of infrared difference bands. These bands correspond to changes in the vibrational modes of individual molecular groups that are structurally active during receptor activation, i.e., during the rhodopsin to metarhodopsin II transition. The orientation of the transition dipole moments of bands previously assigned to the carboxyl (C=O) groups of Asp83 and Glu113 has been determined. The orientation of specific groups in the retinylidene chromophore has been inferred from the dichroism of the bands associated with the polyene C-C, C=C, and hydrogen-out-of-plane vibrations. Interestingly, the use of polarized infrared light reveals several difference bands in the rhodopsin to metarhodopsin II difference spectrum which were previously undetected, e.g., at 1736 and 939 cm(-1). The latter is tentatively assigned to the hydrogen-out-of-plane mode of the HC(11)=C(12)H segment of the chromophore. Our data suggest a significant change in orientation of this group in the late phase of rhodopsin activation. On the basis of available site-directed mutagenesis data, bands at 1406, 1583, and 1736 cm(-1) are tentatively assigned to Glu134. The main features in the amide regions in the dichroic difference spectrum are discussed in terms of a slight reorientation of helical segments upon receptor activation.