Engineering a Rhodopsin Protein Mimic

Abstract

Due to the difficulties in handling and manipulating membrane-bound proteins, such as rhodopsin, and the lack of crystallographic information on the cone opsins, we have opted to engineer a protein mimic of the transmembrane G-protein coupled receptor. Human cellular retinoic acid binding protein (CRABPII), a well studied and characterized protein, has been reengineered into a protein that now will bind retinal as a protonated Schiff base with high binding affinity (Kd = 2 nM) mimicking that of rhodopsin.

The Structure of Apo-wild-type Cellular Retinoic Acid Binding Protein II at 1.4 Å and its Relationship to Ligand Binding and Nuclear Translocation

“…This mutant is still capable of RA binding, though the affinity is significantly reduced (K d = 40(±4) nM) compared to WT-CRABPII (K d = 2.0(±1.2) nM). 52 Overall structure of CRABPII•RA and comparison with apo-WT CRABPII…”

“…The PCR products were transfected into JM109 competent Escherichia coli cells for plasmid maintenance. 52 General procedure for expression and purification of CRABPII clones Human recombinant CRABPII was expressed and purified as described. 47 The target gene (CRABPII/pET17b), as isolated from JM109 using Qiagen's Maxi Prep® DNA isolation kit, was transformed into E. coli strain BL21 (DE3)pLysS cells (Stratagene), according to standard protocols.…”

Structural analysis of site-directed mutants of cellular retinoic acid-binding protein II addresses the relationship between structural integrity and ligand binding

“…The primers and procedure are described in the supporting information of our previously published data (Crist et al, 2006;Vasileiou et al, 2007).…”

Domain-Swapped Dimers of Intracellular Lipid-Binding Proteins: Evidence for Ordered Folding Intermediates

“…(Wang et al, 2012) The robustness of this family of proteins, in particular hCRBPII, to mutations was the central feature that led us to use them as templates to develop rhodopsin mimics for the purpose of investigating factors that contribute to wavelength regulation. (Crist et al, 2006; Huntress et al, 2013; Lee et al, 2012; Nossoni et al, 2014; Vaezeslami et al, 2008; Vaezeslami et al, 2006; Vasileiou et al, 2009a; Vasileiou et al, 2007; Vasileiou et al, 2009b; Wang et al, 2014; Wang et al, 2012; Yapici et al, 2015) During the preparation of mutants targeted for these studies, we observed protein bands that eluted separately on ion-exchange chromatography, but nonetheless, were the same species on SDS-PAGE. This led us to the discovery of a set of hCRBPII protein mutants capable of domain swapped dimerization.…”

“…(Cowan et al, 1993; Nossoni et al, 2014; Wang et al, 2012) Both hCRBPII and other members of this family are remarkably resilient to mutation. (Berbasova et al, 2013; Berbasova et al, 2016; Crist et al, 2006; Huntress et al, 2013; Lee et al, 2012; Nossoni et al, 2014; Vaezeslami et al, 2008; Vaezeslami et al, 2006; Vasileiou et al, 2009a; Vasileiou et al, 2007; Vasileiou et al, 2009b; Wang et al, 2014; Wang et al, 2012; Yapici et al, 2015) For example more than 150 structurally stable mutants of hCRBPII have been characterized in our lab in the course of our studies of protein-chromophore interactions. (Wang et al, 2012) The robustness of this family of proteins, in particular hCRBPII, to mutations was the central feature that led us to use them as templates to develop rhodopsin mimics for the purpose of investigating factors that contribute to wavelength regulation.…”