Development of photoswitchable rhodopsin mimics: Spectroscopy, structural studies and wavelength regulation studies of these systems

Retinal photo pigments have been an essential part of life on the planet from bacteria to mammals. Photo isomerization of the retinal is the heart of the function of all of these pigments. Vision and circadian rhythm in higher organisms and energy production and phototaxis in bacteria are all mediated by these light absorbing proteins. Retinal isomerization has never been recapitulated in an artificial retinal protein pigment before. Reengineering of human Cellular Retinoic Acid Binding ProteinII (hCRABPII) into a photo switchable retinal pigment provided a light absorbing pigment with unique properties. The reengineered protein can isomerize thermodynamically and photochemically from a 15- cis retinal to 15-trans retinal and vice versa. The isomerization is accompanied by a 3-5 unit of pKa changing from above physiological pH to lower similar to other retinal pigments. Isomerization of retinal is quantitative both in solution and more importantly in the crystalline form. High-resolution X-ray crystal structures provide a crystal clear image for the isomerization of the retinal in this system. The quantitative isomerization of the retinal in the crystalline form can be a valuable tool in characterizing the intermediates of retinal isomerization. This protein in combination with fluorescent proteins can be used as a fluorescent quencher to develop a Reversibly Switchable Fluorescent Protein (RSFP).;Wavelength regulation of rhodopsins has been a long standing question with no solid answer that has been debated for five decades. Point charges from charged residues and the dipole moment from the polar residues, especially hydroxyl containing side chains and the conformational changes in the retinal chromophore have been suggested as the main theories for wavelength regulation. However, the effect of the polar water molecules in wavelength regulation of retinal pigments has been less investigated. Using a reengineered human Cellular Retinol Binding ProteinII (hCRBPII), we were able to systematically demonstrate a coherent effect of the polar water molecules in the wavelength regulation of hCRBPII retinal protein complexes, which can be attributed to other known retinal light absorbing pigments as well. Mutating the residues interacting with these water molecules, which are located close to the retinal polyene from the beginning to the end of it, regulate the absorption of the protein.