Conformations of the rhodopsin third cytoplasmic loop grafted onto bacteriorhodopsin

Abstract

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.

G‐protein‐coupled receptor domain overexpression in Halobacterium salinarum: Long‐range transmembrane interactions in heptahelical membrane proteins

“…Cysteine scanning of the cytoplasmic end of helix six also produced minimal perturbation of the transitions between the M, N, and O substates 62. Further, our results are also consistent with other studies investigating loop insertion/deletion mutants, which indicate that the bR loop regions contribute to folding and stability but minimally to bR's photochemical activity 6, 61, 64…”

“…However, portions of α 2b ‐AR 3i produced by cell‐free expression were shown to contain ligand binding determinants 21. We expressed α 2b ‐AR‐bR loop region fusion proteins following strategies developed for expression of bR, hR, GFP, and GPCR in H. salinarum 4, 6, 9, 28, 54, 55 . H. salinarum is a logical choice in which to attempt GPCR expression because it produces high levels of the GPCR ortholog bR, and H. salinarum cells are easy and inexpensive to culture.…”

“…1610). We therefore utilized a domain approach to investigate GPCR structure and function 4–6. We have fused GPCR extramembranous domains into structurally homologous regions of bacteriorhodopsin and expressed the fusion proteins in Halobacterium salinarum 7–9.…”

Sampling effects influence heights measured with atomic force microscopy

“…Such accurate height measurements can be used to scale the volume of an object and define the envelope for a molecular structure. This envelope can be used to interpret structures solved by other methods such as X‐ray and electron microscopy (Schabert et al ., 1995; Heymann et al ., 1999) and to determine conformations of molecular systems (Heymann et al ., 2000; McMaster et al ., 1996, 1999). From our measurements it became clear that the accuracy of AFM height measurements depends on resolving the surface structures of the specimen.…”

Modelling of third cytoplasmic loop of bovine rhodopsin by multicanonical molecular dynamics

“…In these three crystal structures, H8 is located between the seventh TMH (TMH7) and C4, lying parallel to the cytoplasmic membrane. The C3 structure, in particular, has been investigated in various experiments: nuclear magnetic resonance (NMR) spectroscopy [12], spin-labelling studies [13], and atomic force microscopy [14]. The NMR study showed that the C3 fragment formed a turn-helix-turn conformation.…”