Negative stain electron microscopy and saturation transfer electron spin resonance spectroscopy have been used to compare the lattice ordering and in-plane membrane mobility of full-length and C-terminally cleaved squid rhodopsin. The Cterminus of squid rhodopsin contains a negatively charged region followed by 9-10 repeats of a proline-rich sequence, not found in rhodopsins other than those of cephalopod invertebrates, but similar proline repeats are found in other, unrelated membrane proteins. We find that the proline repeats cluster the rhodopsins into small groups, interfering with two-dimensional crystallization and maintaining their mobility in the membrane.Key words." 2D crystal; Negative stain electron microscopy; Spin label ESR (spectroscopy); Rotational diffusion; Cephalopod rhodopsin spectroscopy on squid rhodopsin with and without the C-terminal extension, purified and reconstituted after proteolytic cleavage. Unexpectedly, the rotational correlation time of squid rhodopsin increased by a further 2-fold after cleavage and membrane reassembly. The mobility measurements agree well with electron microscopy (EM) studies showing ordered array formation and an increase in crystallinity with increasing purification.This work shows that the 40 kDa 'core' of squid rhodopsin contains interactive regions that cause ordering of rhodopsin monomers into larger, less mobile assemblies. The presence of the C-terminal extension appears to limit the array formation.

The biochemical role of the visual-pigment protein, rhodopsin, is reviewed, with reference to vertebrate rods and cones and the microvillar photoreceptors of invertebrates. New results are presented on the structure of squid rhodopsin, which possesses an extensive proline-rich repeat at its C-terminus, using negative-stain electron microscopy.