ABSTRACT
Protein-chromophore interactions are a central component of a wide variety of critical biological processes such as color vision and photosynthesis. To understand the fundamental elements that contribute to spectral tuning of a chromophore inside the protein cavity, we redesigned human cellular retinol binding protein II (hCRBPII) to fully encapsulate all-trans-retinal and form a covalent bond as a protonated Schiff base. This system, using rational mutagenesis designed to alter the electrostatic environment within the binding pocket of the host protein, enabled regulation of the absorption maximum of the pigment in the range of 425 to 644 nanometers. With only nine point mutations, the hCRBPII mutants induced a systematic shift in the absorption profile of all-trans-retinal of more than 200 nanometers across the visible spectrum.
Subject(s)
Retinaldehyde/analogs & derivatives , Retinol-Binding Proteins, Cellular/chemistry , Absorption , Electrons , Humans , Mutagenesis , Point Mutation , Retinaldehyde/chemistry , Retinol-Binding Proteins, Cellular/genetics , Static ElectricityABSTRACT
Cellular Retinoic Acid Binding Protein II (CRABPII) has been reengineered to specifically bind and react with all-trans-retinal to form a protonated Schiff base. Each step of this process has been dissected and four residues (Lys132, Tyr134, Arg111, and Glu121) within the CRABPII binding site have been identified as crucial for imine formation and/or protonation. The precise role of each residue has been examined through site directed mutagenesis and crystallographic studies. The crystal structure of the R132K:L121E-CRABPII (PDB-3I17) double mutant suggests a direct interaction between engineered Glu121 and the native Arg111, which is critical for both Schiff base formation and protonation.
