Fig. 6.

Functional characterization of cellular retinol binding protein 2 (CRBP2)^T51I/V62M mutant. A: Sequencing of mutated CRBP2 cDNA indicated the intended substitutions. The right panel represents mass spectrum of intact CRBP2^T51I/V62M. The deconvoluted molecular weight of this protein was identical to that calculated based on the amino acid sequence. B: UV/visible absorbance spectra of WT and mutated CRBP2. The spectral characteristic between 330 and 380 nm indicated formation of the protein-atROL complex. C: Location of the mutated residues within the binding cavity of cellular retinol binding protein 1 (gray) and CRBP2 (pink). Residues I51 and M62 present in the cellular retinol binding protein 1 are colored orange. 2-Arachidonoylglycerol (2-AG) molecule present in the binding pocket is represented by ball and stick. D: Fluorescence spectra from CRBP2^T51I/V62M titrated with increasing concentrations of 2-AG. Direction of the changes in the fluorescence signal for the protein scaffold and the retinoid moiety are marked with green and red arrows, respectively. The titration curves for WT and CRBP2^T51I/V62M variant reveal a dramatic shift in the K_d values for the mutated protein (inset).