Binding of transducin and transducin-derived peptides to rhodopsin studies by attenuated total reflection-Fourier transform infrared difference spectroscopy.
Fourier transform infrared difference spectroscopy combined with the attenuated total reflection technique allows the monitoring of the association of transducin with bovine photoreceptor membranes in the dark. Illumination causes infrared absorption changes linked to formation of the light-activated rhodopsin-transducin complex. In addition to the spectral changes normally associated with meta II formation, prominent 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 becomes protonated upon formation of the activated complex. Reconstitution of rhodopsin into phosphatidylcholine vesicles has little influence on the spectral properties of the rhodopsin-transducin complex, whereas pH affects the intensity of the carbonyl stretching band. AC-terminal peptide comprising amino acids 340-350 of the transducin alpha-subunit reproduces the frequencies and isotope sensitivities of several of the transducin-induced bands between 1500 and 1800 cm-1, whereas an N-terminal peptide (aa 8-23) does not. Therefore, the transducin-induced absorption changes can be ascribed mainly to an interaction between the transducin-alpha C-terminus and rhodopsin. The 1735 cm-1 vibration is also seen in the complex with C-terminal peptides devoid of free carboxylic acid groups, indicating that the corresponding carbonyl group is located on rhodopsin.
Fahmy
K
K
Institut für Biophysik und Strahlenbiologie der Albert-Ludwigs-Universität Freiburg, Germany. fahmy@ruf.uni-freiburg.de
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002417:Cattle; D017666:Deuterium Oxide; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008081:Liposomes; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010713:Phosphatidylcholines; D010777:Photochemistry; D020419:Photoreceptor Cells, Vertebrate / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D012243:Rhodopsin / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D015293:Transducin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008081:Liposomes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D012243:Rhodopsin; D015293:Transducin; D017666:Deuterium Oxide