Regulation of Absorption and Emission in a Protein/Fluorophore Complex

Abstract

Human cellular retinol binding protein II (hCRBPII) was used as a protein engineering platform to rationally regulate absorptive and emissive properties of a covalently bound fluorogenic dye. We demonstrate the binding of a thio-dapoxyl analog via formation of a protonated imine between an active site lysine residue and the chromophore's aldehyde. Rational manipulation of the electrostatics of the binding pocket results in a 204 nm shift in absorption and a 131 nm shift in emission. The protein is readily expressed in mammalian systems and binds with exogenously delivered fluorophore as demonstrated by live-cell imaging experiments.

Figure 1

Spectroscopic properties of the free aldehyde TD-1V and its corresponding PSB formed with n-butyl amine in a variety of solvents. (a) Absorbance and; (b) emission of the free aldehyde; (c) absorbance; and (d) emission of the TD-1V-PSB with n-butyl amine. Note, the emission values for the PSB in THF were 10 times larger than that shown on the plot.

Figure 2

Crystal structure of M11/TD-1V and space-filling representation. Electron density is shown at 1σ.

Figure 3

(a) Crystal structure of M11/TD-1V, highlighting the water mediated network from Y19 to TD-1V. (b). Crystal structure of M14/TD-1V shows this water network is abolished.

Figure 4

(a) Residues mutated (shown in green) to encapsulate the binding cavity. Crystal structure is of M14/TD-1V. (b) Positions for introducing an acidic reside to blue shift wavelength based on the crystal structure of M11/TD-1V.

Figure 5

(a) Range of absorption and (b) emission achieved in this study by coupling hCRBPII monomers with TD-1V. Mutants from left to right are M32, M27, M28, M10, M8, M17, and M18 (M25–M34, Table S8); (c) plot of emission versus absorbance for the approximately 80 hCRBPII/TD-1V complexes examined in this study yields a linear fit, with R² = 0.93.

Figure 6

Labeling of HeLa cells expressing M22-EGFP, M22-EGFP-3NLS and M22-EGFP-NES with 10 μM TD-1V (incubated at 37 °C for 1 h). NLS = nuclear localization sequence and NES = nuclear export signal.