UC San Diego

The green alga Chlamydomonas reinhardtii has a phototaxis response time less than 50usec, suggesting that the photoreceptor and channel form a protein complex or are a single protein. I extracted and cloned two genes described as opsin type proteins from C. reinhardtii algae. When heterologously expressed functional channels in HEK293 cells as well as neurons, one of these channels conducted inward currents upon 477nm illumination. Photogeneration of singlet oxygen is a viable mechanism for electronmicroscopic localization and acute inactivation of biarsenical-tagged proteins. Attachment of multiple fluorophores to a single protein would increase sensitivity if proximity-induced quenching could be avoided. I found that a GFP, used as a rigid scaffold molecule, could be decorated with two GFP, used as a rigid scaffold molecule, could be decorated with two tetracysteine motifs, inserted so that the tetracysteines were not palmitoylated, the GFP retained both free termini, and the bound ReAsH fluorophores maintained full fluorescence and singlet oxygen generation