Signal transduction usually involves the binding of signaling molecules, ligands to the receptors. Herein, we will explore signal transduction in vision, a family of GPCR, and, more specifically, its rhodopsin subfamily, which plays a crucial role in color vision and sensing. There have been many mutagenesis studies done using Raman spectroscopy, crystallography, and NMR to understand the mechanism of the wavelength regulation and the photoisomerization of rhodopsin proteins. Moreover, recent studies have successfully indicated the intermediates of rhodopsin's photocycle, using time-resolved experiments, femtosecond x-ray laser (x-ray free-electron laser, XFEL), and cryokinetic data. All of these studies demonstrate the different important biophysical characteristics of rhodopsin systems; however, they have some limitations. Rhodopsins are membrane proteins, and their expression, purification, mutagenesis, and crystallization are very challenging. Also, these proteins evolve a lot during evolution. As a result of environmental changes and developments during evolution, many amino acid residues in rhodopsins become conserved residues; therefore, it is hard to illustrate every single residue's effect on wavelength tuning through mutagenesis studies. Therefore, we use Cellular Retinoic Acid Binding Protein II (CRABPII) and Cellular Retinol Binding Protein II (CRBPII) as mimics to study rhodopsin systems. Their solubility, small size, substantial binding pocket and ease of crystallization makes them a great candidate for our purpose. By using high resolution X-ray crystallography and spectroscopy, we were successful in mimicking wavelength tuning as well as photoisomerization cycle in rhodopsin mimic templates. Another application of these templates is designing new fluorescent dyes. Many fluorophores have been designed based on hCRBPII template in collaboration with Prof. Borhan's group to reach the FarRed-NearIR emission. We postulate mechanisms of these new fluorophores using our structural analysis.During our studies on hCRBPII, my lab-mates characterized the domain swapped dimer as a folding product for this protein. In domain swapping, two or more monomers exchange an identical part of their structures to form a dimer or higher-order oligomer. Almost all of the studies on DSD hCRBPII have been done through bacterial expression. To find out the physiological relevancy of this phenomenon, we tried to investigate the existence of the DSD form in mammalian expression. Also, Since the existence of domain swapping for hCRBPII is likely to have physiological importance, we investigate the mechanism of domain swap dimerization in the other members of iLBP family. We characterized the Domain swapped dimer for WT-human fatty acid binding protein 5 (hFABP5) bound to palmitic acid as a natural product during the E. coli expression. The existence of Domain swapping in FABP5 as another member of the iLBP family is another reason that indicates the formation of DSD as a natural kinetic product during the folding process, which may indicate a common folding pathway for these two proteins.
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