Retinal photo pigments have been an essential part of life on the planetfrom bacteria to mammals. Photo isomerization of the retinal isthe heart of the function of all of these pigments. Vision and circadian rhythmin higher organisms and energy production and phototaxis in bacteria are allmediated by these light absorbing proteins. Retinalisomerization has never been recapitulated in an artificial retinal proteinpigment before. Reengineering of human Cellular Retinoic Acid BindingProteinII (hCRABPII) into a photo switchable retinal pigment provided a lightabsorbing pigment with unique properties. The reengineered proteincan isomerize thermodynamically and photochemically from a 15-cis retinal to15-trans retinal and vice versa. The isomerization is accompanied by a 3-5 unitof pKa changing from above physiological pH to lower similar to other retinal pigments.Isomerization of retinal is quantitative both in solution and more importantly inthe crystalline form. High-resolution X-ray crystal structures provide a crystalclear image for the isomerization of the retinal in this system. The quantitativeisomerization of the retinal in the crystalline form can be a valuable tool incharacterizing the intermediates of retinal isomerization. This protein incombination with fluorescent proteins can be used as a fluorescent quencherto develop a Reversibly Switchable Fluorescent Protein (RSFP).Wavelength regulation of rhodopsins has been a long standing question withno solid answer that has been debated for five decades. Point charges fromcharged residues and the dipole moment from the polar residues, especiallyhydroxyl containing side chains and the conformational changes in the retinalchromophore have been suggested as the main theories for wavelengthregulation. However, the effect of the polar water molecules in wavelengthregulation of retinal pigments has been less investigated. Using areengineered human Cellular Retinol Binding ProteinII (hCRBPII), we wereable to systematically demonstrate a coherent effect of the polar watermolecules in the wavelength regulation of hCRBPII retinal protein complexes,which can be attributed to other known retinal light absorbing pigments aswell. Mutating the residues interacting with these water molecules, which arelocated close to the retinal polyene from the beginning to the end of it,regulate the absorption of the protein.
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