title: Conformations of the rhodopsin third cytoplasmic loop grafted onto bacteriorhodopsin.
abstract: BACKGROUND
  The third cytoplasmic loop of rhodopsin (Rho EF) is important in signal transduction from the retinal in rhodopsin to its G protein, transducin. This loop also interacts with rhodopsin kinase, which phosphorylates light-activated rhodopsin, and arrestin, which displaces transducin from light-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 surfaces of purple membrane containing the mutant BR (called IIIN) were imaged by atomic force microscopy (AFM) under physiological conditions to a resolution of 0.5-0.7 nm. The crystallinity and extracellular surface of IIIN were not perturbed, and the cytoplasmic surface of IIIN increased in height compared with BR, consistent with the larger loop. Ten residues of Rho EF were 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 the AFM data of IIIN was used to select probable models.
  
  
  CONCLUSIONS
  A likely conformation of Rho EF involves some extension of helices E and F, with the tip of the loop lying over helix C and projecting towards 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.
authors.affiliation: M.E. Müller-Institute for Structural Biology, Biozentrum, University of Basel, Basel, CH-4056, Switzerland.
authors.forename: J B
authors.initials: JB
authors.lastname: Heymann
authors.forename: M
authors.initials: M
authors.lastname: Pfeiffer
authors.forename: V
authors.initials: V
authors.lastname: Hildebrandt
authors.forename: H R
authors.initials: HR
authors.lastname: Kaback
authors.forename: D
authors.initials: D
authors.lastname: Fotiadis
authors.forename: B
authors.initials: B
authors.lastname: Groot
authors.forename: A
authors.initials: A
authors.lastname: Engel
authors.forename: D
authors.initials: D
authors.lastname: Oesterhelt
authors.forename: D J
authors.initials: DJ
authors.lastname: Müller
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D002417:Cattle; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure
chemical_list: D011994:Recombinant Proteins; D001436:Bacteriorhodopsins; D012243:Rhodopsin