Conformations of the rhodopsin third cytoplasmic loop grafted onto bacteriorhodopsin

Background: The third cytoplasmic loop of rhodopsin (Rho EF) is important i n signal transduction from the retinal in rhodopsin to its G protein, trans ducin. This loop also interacts with rhodopsin kinase, which phosphorylates light-activated rhodopsin, and arrestin, which displaces transducin from l ight-activated phosphorylated rhodopsin. Results: We replaced eight residues of the EF loop of bacteriorhodopsin (BR ) with 24 residues from the third cytoplasmic loop of bovine Rho EF. The su rfaces of purple membrane containing the mutant BR (called IIIN) were image d by atomic force microscopy (AFM) under physiological conditions to a reso lution of 0.5-0.7 nm. The crystallinity and extracellular surface of IIIN w ere not perturbed, and the cytoplasmic surface of IIIN increased in height compared with BR, consistent with the larger loop. Ten residues of Rho EF w ere excised by V8 protease, revealing helices E and F in the AFM topographs . Rho EF was modeled onto the BR structure, and the envelope derived from t he AFM data of IIIN was used to select probable models. Conclusions: A likely conformation of Rho EF involves some extension of hel ices E and F, wit-h the tip of the loop lying over helix C and projecting t owards the C terminus. This is consistent with mutagenesis data showing the TTQ transducin-binding motif close to loop CD, and cysteine cross-linking data indicating the C-terminal part of Rho EF to be close to the CD loop.