Engineering a functional blue-wavelength-shifted rhodopsin mutant

We report an effort to engineer a functional, maximally blue-wavelength-shi fted version of rhodopsin. Toward this goal, we first constructed and assay ed a number of previously described mutations in the retinal binding pocket of rhodopsin, G90S, E122D, A292S, and A295S. Of these mutants, we found th at only mutants E122D and A292S were like the wild type (WT). In contrast, mutant G90S exhibited a perturbed photobleaching spectrum, and mutant A295S exhibited decreased ability to activate transducin. We also identified and characterized a new blue-wavelength-shifting mutation (at site T118), a re sidue conserved in most opsin proteins. Interestingly, although residue T11 8 contacts the critically important C-9-methyl group of the retinal chromop hore, the T118A mutant exhibited no significant perturbation other than the blue-wavelength shift. In analyzing these mutants, we found that although several mutants exhibited different rates of retinal release, the activatio n energies of the retinal release were all similar to 20 kcal/mol, almost i dentical to the value found for WT rhodopsin. These latter results support the theory that chemical hydrolysis of the Schiff base is the rate-limiting step of the retinal release pathway. A combination of the functional blue- wavelength-shifting mutations was then used to generate a triple mutant (T1 18A/E122D/A292S) which exhibited a large blue-wavelength shift (absorption lambda (max) = 453 nm) while exhibiting minimal functional perturbation. Mu tant T118A/E122D/A292S thus offers the possibility of a rhodopsin protein t hat can be worked with and studied using more ambient lighting conditions, and facilitates further study by fluorescence spectroscopy.