I. The intrinsic mechanism for wavelength regulation observed in color rhodopsins has been under intense study since the last century. One single chromophore, retinal, was found to be absorb over a wide range of the visible spectrum, from 420 nm to 560 nm, depending on which rhodopsin it is bound to. Different model compound studies, rhodopsin mutagenesis studies and computational studies have been carried out to understand what causes the spectral differences. However, this question is still not conclusively answered, due to lack of crystal structures of the color rhodopsins and rhodopsin mutants that were made for wavelength regulation studies. Our lab has engineered a small cellular protein, Cellular Retinoic Acid Binding Protein II, into a rhodopsin mimic that can bind all-trans-retinal as a protonated Schiff base. Further studies demonstrated that full sequestration of the chromophore from the bulk solvent is critical for spectral tuning. Therefore, a second generation rhodopsin mimic using Cellular Retinol Binding Protein II (CRBPII) was engineered and mutagenesis was carried out to study the cause-effect relationships of different stereo-electronic effects on wavelength regulation. We were able to regulate the wavelength over an unprecedented range, from 474 nm to 644 nm, surpassing the existing limits. Electrostatic calculations based on the high resolution crystal structures of CRBPII mutants revealed that electrostatic interactions are playing the major role in the spectral tuning observed.II. Fluorescent protein tags have been widely applied in microbiological studies to study the protein expression level, protein localization, protein-protein interactions and some important biological events. GFP and GFP like proteins have been greatly developed, along with some other fluorescent protein tags. However, in the fluorescence palette, bright photostable red fluorescent and near-IR are still lacking. Due to the robustness of CRBPII mutants, we wanted to functionalize them into red fluorescent protein tags or near-IR fluorescent protein tags by using appropriate chromophores. One chromophore, Mero-1, a merocyanine analogue of retinal, has proved to be suitable to be used along with CRBPII mutants as red fluorescent protein tags both in prokaryotic and eukaryotic systems.III. Azobenzene has been widely applied in material science and chemical biological systems, in order to achieve photoswitchable properties. We want to design a protein tag that can bind specifically to the trans-isomer of the azobenzene derivatives. Upon light irradiation, the azobenzene derivative will isomerize to cis. Consequently, the protein tag will lose its affinity to the cis-isomer and dissociate. This photoswitchable protein tag can be used for light-controlled protein purification. Phage display was applied to evolve this protein tag from the phage library generated based on WT-CRABPII.
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