Abstract

Asp83 is a highly conserved residue in the second transmembrane domain of visual pigments and many members of other G proteincoupled receptor subfamilies. Upon illumination, the rod visual pigment rhodopsin proceeds through various intermediate states (Batho BSI Lumi Meta I Meta II). Meta II represents the active state of rhodopsin, which binds and activates the G protein transducin. Evidence has been presented that Asp83 participates in the formation of Meta II and undergoes a change in Hbonding. To investigate whether this role of Asp83 requires its protondonating capacity and/or its Hbonding capability, we constructed the mutants D83C and D83N. Both mutants appear to effectively activate transducin, indicating that Asp83 is not essential for signal transduction. Differential effects of the mutations D83C and D83N are observed in the spectral properties and the pH sensitivity of the Meta I Meta II transition. In general, D83C behaves much more like wildtype than D83N. We conclude that the structural role of Asp83 also involves the acidic nature of its carboxyl group. In addition, the participation in Meta II formation of Cys83 in D83C manifests itself as a change in the vibrational properties of the sulfhydryl group, demonstrating that the SH group can be used as a noninvasive probe for local structural changes.