A structural role for Asp83 in the photoactivation of rhodopsin

Asp83 is a highly conserved residue in the second transmembrane domain of v isual pigments and many members of other G protein-coupled receptor subfami lies. Upon illumination, the rod visual pigment rhodopsin proceeds through various intermediate states (Batho <----> BSI --> Lumi --> Meta I <----> Me ta II). Meta II represents the active state of rhodopsin, which binds and a ctivates the G protein transducin. Evidence has been presented that Asp83 p articipates in the formation of Meta II and undergoes a change in H-bonding . To investigate whether this role of Asp83 requires its proton-donating ca pacity and/or its H-bonding capability, we constructed the mutants D83C and D83N. Both mutants appear to effectively activate transducin, indicating t hat Asp83 is not essential for signal transduction. Differential effects of the mutations D83C and D83N are observed in the spectral properties and th e pH sensitivity of the Meta I --> Meta II transition. In general, D83C beh aves much more like wild-type than D83N. We conclude that the structural ro le of Asp83 also involves the acidic nature of its carboxyl group. In addit ion, the participation in Meta II formation of Cys83 in D83C manifests itse lf as a change in the vibrational properties of the sulfhydryl group, demon strating that the -SH group can be used as a non-invasive probe for local s tructural changes.