1) PROBING THE WAVELENGTH REGULATION OF RHODOPSIN PIGMENTS VIA DE NOVO PROTEIN ENGINEERING OF A RHODOPSIN MIMICAfter several decades of research, the mechanism of wavelength regulation in human rhodopsins has not been fully elucidated at a molecular level. Although the gene sequences of color rhodopsin (Blue, Green and Red) and Rod rhodopsin have been released and the crystal structure of bovine rhodopsin has been resolved, the important protein-ligand interactions that lead to wavelength modulation have not been dissected in details. In order to tackle the problems arisen using rhodopsin to study the theories behind wavelength regulation, a de novo approach has been taken by engineering a protein surrogate as a rhodopsin mimic to bind all- trans-retinal as a protonated Schiff base. Throughout the study with cellular retinoic acid binding protein II (CRABPII) as a rhodopsin mimic, we discovered that a fully embedded chromophore is needed for the system to respond to amino acid changes. Therefore, a shorter chromophore, C15 aldehyde, has been used instead of a full-length all-trans-retinal. In addition, based on the electrostatic potential calculations on color opsins models and the rhodopsin crystal structure, we hypothesize that the electrostatic potential projected on the bound chromophore from the protein plays an important role on wavelength regulation. To investigate the hypothesis, the crystal structure of C15 aldehyde with CRABPII mutant R132K:R111L:L121E:R59W was used as a template and a series of mutants has been generated and tested. We are able to modulate the absorption maxima of the bound C15 aldehyde PSB from 380 nm to 424 nm based on the change of the overall electrostatic potential projected on the bound chromophore by the protein. 2) ENGINEERING CRABPII AS A RETINAL ISOMERASE AND A PROTEIN FUSION TAG Protein engineering has been developed for decades to solve the problem in chemistry, biomedical research, pharmaceutical and medical industry. In human eyes, upon absorption of a photon, isomerization of 11-cis-retinal specifically to all-trans-retinal leads to rhodopsin conformational changes which induce signal transduction and allow us to see. Rhodopsin has a high affinity to 11-cis-retinal and poor binding to all-trans-retinal which allows rhodopsin to reload with 11-cis-retinal and the vision cycle continues. We would like to apply the same principle of design to re-engineer CRABPII into retinal isomerase upon absorption of specific wavelength. During the process of re-engineering CRABPII into a retinal isomerase, we found that CRABPII has an ability to isomerize 11-cis-retinal to all-trans-retinal without irridation with light. We, therefore, conduct a series of experiments to study the mechanism of isomerization and propose that the isomerization is the result of conjugated addition of a nucleophile, rotation and elimination of the nucleophile. In addition, our study suggested that the ordered water in the binding cavity plays a vital role in this retinal isomerization in dark. Recently, protein fusion tags play an important role in biological research for their ability to probe the biological processes in Nature. Green fluorescent protein (GFP) is one of the commonly used tags. However, it is not without limitations; for example, it only works in aerobic condition. In order to provide a protein fusion tag that is orthogonal to GFP, we would like to re-engineer CRABPII as a fluorescent protein through incorporation of suitable chromophores. We have successfully re-engineered CRABPII to bind to a merocyanine as a protonated Schiff base. The resulting protein-merocyanine complex is red shifted to ~590 nm from 465 nm and fluoresces at ~615 nm with the quantum yield ~10%. In addition, we are able to modulate the absorption and fluorescence of bound merocyanine (λmax from 560 to 598 nm, λem from 604 nm to 615 nm) through mutagenesis.
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