Probing rhodopsin-transducin interactions by surface modification and mass spectrometry.
The interactions of rhodopsin and the alpha-subunit of transducin (G(t)) have been mapped using a surface modification "footprinting" approach in conjunction with mass spectrometric analysis employing a synthetic peptide corresponding to C-terminal residues 340-350 of the alpha-subunit of G(t), G(t)alpha(340-350). Membrane preparations of unactivated (Rh) and light-activated rhodopsin (Rh*), each in the presence or absence of G(t)alpha(340-350), were acetylated with the water-soluble reagent sulfosuccinimidyl acetate, and the extent of the acetylation was determined by mass spectrometry. By comparing the differences in acetylation among Rh, Rh*, and the Rh-G(t)alpha(340-350) and Rh*-G(t)alpha(340-350) complexes, we demonstrate that the surface exposure of the acetylation sites was reduced by the conformational change associated with light activation, and that binding of G(t)alpha(340-350) blocks acetylation sites on cytoplasmic loops 1, 2, and 4 of Rh*. In addition, we show evidence of interaction between the end of the C-terminal tail of rhodopsin and G(t)alpha in the unactivated state of rhodopsin.
Wang
Xin
X
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Kim
Sung-Ho
SH
Ablonczy
Zsolt
Z
Crouch
Rosalie K
RK
Knapp
Daniel R
DR
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D003593:Cytoplasm / Q000378:metabolism; D008027:Light; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013499:Surface Properties; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism*
D010446:Peptide Fragments; D021122:Protein Subunits; D012243:Rhodopsin; D015293:Transducin; D008239:Lysine