Excerpts

Here, we describe frFAST, a small monomeric protein tag of 14 kDa that forms a far-red fluorescent assembly with (4- hydroxy-3-methoxy-phenyl)allylidene rhodanine (HPAR- 3OM) (Figure 1 a) and enables the observation of fusion proteins in live cells and organisms. frFAST is a variant of FAST (Fluorescence-Activating and absorption-Shifting Tag), a monomeric protein tag engineered from the apo photoactive yellow protein (PYP) from Halorhodospira halophila.

Here, we describe frFAST, a small monomeric protein tag of 14 kDa that forms a far-red fluorescent assembly with (4- hydroxy-3-methoxy-phenyl)allylidene rhodanine (HPAR- 3OM) (Figure 1 a) and enables the observation of fusion proteins in live cells and organisms. frFAST is a variant of FAST (Fluorescence-Activating and absorption-Shifting Tag), a monomeric protein tag engineered from the apo photoactive yellow protein (PYP) from Halorhodospira halophila....The resulting variant possesses the mutations F62L, D71V, P73S, E74G and V107I relative to FAST. F62L and V107I are localized in the chromophore binding site, while D71V, P73S, E74G are in close proximity with the chromophore entry site.... This optimized variant binds HPAR-3OM tightly with a KD of 1 mm (Figure S3), and strongly activates its far-red fluorescence (Figure 1 b), form- ing a fluorescent complex with 555 nm/670 nm absorption- emission peaks, f = 21 %, and a molar absorptivity e = 45 000 m@1 cm@1. HPAR-3OM undergoes a 130 nm red shift in absorption upon binding,