BINDING OF TRANSDUCIN AND TRANSDUCIN-DERIVED PEPTIDES TO RHODOPSIN STUDIED BY ATTENUATED TOTAL REFLECTION-FOURIER TRANSFORM-INFRARED DIFFERENCE SPECTROSCOPY

Fourier transform infrared difference spectroscopy combined with the a ttenuated total reflection technique allows the monitoring of the asso ciation of transducin with bovine photoreceptor membranes in the dark. Illumination causes infrared absorption changes linked to formation o f the light-activated rhodopsin-transducin complex. In addition to the spectral changes normally associated with meta II formation, prominen t absorption increases occur at 1735 cm(-1), 1640 cm(-1), 1550 cm(-1), and 1517 cm(-1). The D2O sensitivity of the broad carbonyl stretching band around 1735 cm(-1) indicates that a carboxylic acid group become s protonated upon formation of the activated complex. Reconstitution o f rhodopsin into phosphatidylcholine vesicles has little influence on the spectral properties of the rhodopsin-transducin complex, whereas p H affects the intensity of the carbonyl stretching band. A C-terminal peptide comprising amino acids 340-350 of the transducin cu-subunit re produces the frequencies and isotope sensitivities of several of the t ransducin-induced bands between 1500 and 1800 cm(-1), whereas an N-ter minal peptide (aa 8-23) does not. Therefore, the transducin-induced ab sorption changes can be ascribed mainly to an interaction between the transducin-alpha C-terminus and rhodopsin. The 1735 cm(-1) vibration i s also seen in the complex with C-terminal peptides devoid of free car boxylic acid groups, indicating that the corresponding carbonyl group is located on rhodopsin.