The field of protein engineering has undergone phenomenal growth from its inception approximately 20 years ago. A wide variety of topics have been addressed, including the construction of new protein folds, the introduction of metal binding sites that are both structural and catalytic, the development of novel enzymatic activity, and the creation and optimization of new ligand binding sites. However, left behind has been the issue of protein/chromophore interactions that regulate the spectroscopic properties of bound chromophores. To understand the fundamental elements that contribute to spectral tuning of a chromophore inside a protein cavity, we redesigned small cyctosolic human proteins to fully encapsulate retinylidene based fluorescent and/or nonfluorescent ligands, bound as a protonated Schiff base. Rational mutagenesis, designed with the goal to alter the electrostatic environment within the binding pocket of the host protein, enabled regulation of the absorption and emission spectra of the protein/chromophores complexes.An initial aim of this project was to reengineer Cellular Acid Binding Protein II (CRABPII) into a fluorescent protein via coupling with a nonfluorescent cyanine dye precursor. The designed precursor, merocyanine aldehyde, is capable of binding to variety of CRABPII mutants, resulting in stable fluorescent complexes. In the course of this study, it became evident that CRABPII/merocyanine complexes demonstrate structural variety in terms of ligand orientation and geometry that correlates well with the observed fluorescent properties.From the point of view of structural chemical biology, the next challenge in the field would be to obtain detailed insights on wavelength changes with respect to chromophore modifications. We have focused on coupling of the reengineered hCRBPII rhodopsin mimics with different retinal analogs in order to systematically study the role of structural variations of these ligands on spectral tuning of the resultant pigments. Diverse sets of hCRBPII-retinoid complexes were evaluated with respect to their photophysical properties in light of available high resolution crystal structures. Given the results reported here, we highlight the significance of ring methyl substituents in wavelength regulation, not only as a handle for conformational issues, but also surprisingly as modulators for electrostatic interactions.In this report, we also aimed at designing a protein system that couples with an azobenzene-based chromophore to respond to light stimulation. A synthetic azo chomophore, Azo-AA, was tested with a variety of hCRBPII mutants for affinity optimization. These protein/Azo-AA hybrids could be further engineered for studying protein-protein interactions and generating a bio-machinery for affinity-based protein separation methodology.
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