Figure 2.

Engineering FRET sensors for measuring retinal entry and release kinetics

(A) The assay for measuring retinal entry and release kinetics. The purified Rho is reconstituted in lipidic bicelles.

(B) Design of FRET sensors utilizing the intrinsic Trp fluorescence and extrinsic fluorophore reporter. The location of five Trp residues (blue) and S144 (green) relative to the 11-cis-retinylidene group (orange) in Rho (PDB: 1U19).

(C) Bioorthogonal, site-specific labeling of S144 with strain-promoted alkyne-azide cycloaddition (SpAAC).

(D) The energy transfer scheme for Trp-based and Alexa 488-based assay. Note that energy transfer occurs between Trp and dark-state as well as Meta-II Rho, but is specific to dark-state Rho in the case of Alexa 488.

(E) Representative trace of Trp-based FRET assay.

(F) Representative trace of Alexa 488-based FRET assay.