Reprogramming the biological systems to perform a process other than that dictated by nature illustrates the power of the modern synthetic biology. Protein engineering represents one of the research areas in this field. Crystal structure guided mutagenesis studies allow for a rational protein redesign. Utilizing this de novo approach, Cellular Retinoic Acid Binding Protein II (CRABPII) has been reengineered into a Schiff base forming protein. The reaction is performed deep inside the protein cavity and fascilitates a complete ligand closure from the aqueous media.An initial aim of this project was to design a functioning rhodopsin mimic from CRABPII for the wavelength regulation studies. The strategy combined the principles we learned in the earlier work on CRABPII and human Cellular Retinol Binding Protein II (hCRBPII). The rational modifications of the CRABPII binding cavity provides the effective charge delocalization along the protein-embedded all- trans-retinal-PSB that leads to the regulation of absorption. Reengineered CRABPII is capable of spanning the visible spectrum in the range of 474 - 640 nm. Additionally, CRABPII mutants exhibit an extraordinary range of the iminiumpKa values, ranging from 1.8 to 8.1. The latter phenomena allowed for thechromophoric pH-probe design utilizing the protein-bound chromophoric aldehyde, an isoform of Vitamin A. Moreover, the fusion of the two selected pigments provides a ratiometric protein based pH-sensor.The aldehydes other than retinal might yield alternative pH-sensing ligands with the engineered CRABPII and hCRBPII mutants. With fluorescence being the most widely used detection method in biology, fluorescent and fluorogenic retinal analogs are the primary targets in the ligand redesign. Synthetic julolidine retinal analog meets most of the criteria for an effective fluorogenic probe with pH- sensing properties upon PSB formation. In the protein-bound form this chromophore accounts for the imine-iminium equilibrium with changes in fluorescence intensity. Only iminium yields a light emitting pigment. Moreover, the wavelength shift in response to the pH changes provided the platform for a single protein ratiometric pH-probe design. This pH-dependent shift of absorption is a result of the more effective charge delocalization along the chromophore after protonation of the anionic carboxylate residue in the iminium region.Merocyanine retinal analog shows bright fluorescence and penetrates cells easily. These are valuable criteria for live-cell imaging applications. Optimization of the reaction half-life between hCRBPII mutants and merocyanine retinal analog provided an hCRBPII-tag with instantaneous red fluorescence. Designed method comprises a unique fluorescence recovery after the photobleaching when the cells are treated again with merocyanine.
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