Rational Design of a Colorimetric pH Sensor from a Soluble Retinoic Acid Chaperone

Abstract

Reengineering of cellular retinoic acid binding protein II (CRABPII) to be capable of binding retinal as a protonated Schiff base is described. Through rational alterations of the binding pocket, electrostatic perturbations of the embedded retinylidene chromophore that favor delocalization of the iminium charge lead to exquisite control in the regulation of chromophoric absorption properties, spanning the visible spectrum (474–640 nm). The pKa of the retinylidene protonated Schiff base was modulated from 2.4 to 8.1, giving rise to a set of proteins of varying colors and pH sensitivities. These proteins were used to demonstrate a concentration-independent, ratiometric pH sensor.

Light‐Activated Reversible Imine Isomerization: Towards a Photochromic Protein Switch

“…Note that the imine is protonated at pH 5 and below (p K a =6.6). [12] The only acidic residue with its side chain pointing into the interior of the binding cavity is Glu73. One can surmise that the protonation of Glu73 could reduce the barrier to the hydration of the iminium species.…”

“…Interestingly, all E73A mutants investigated exhibit low iminium p K a values. [12] Putatively, the absence of Glu73 reduces the barrier for isomerization such that the trans -imine is the only observable species.…”

“…[12] The absorption extinction coefficient ( ε ) for R111K:R132L:Y134F:T54V:R59W-CRABPII was reported previously (25038M −1 cm −1 ). [12] …”

“…As described below, our recent efforts in generating rhodopsin protein mimics [11] have also provided the opportunity to produce engineered proteins that recapitulate the change in the p K a of the iminium functionality as a function of isomerization. [12] Here we disclose a protein system that is capable of light-activated isomerization, with concomitant changes to the p K a of the PSB. Specifically, we report for the first time a rhodopsin mimic that can be reversibly photocycled between a protonated cis -iminium species and its deprotonated trans -imine with >4 p K a unit change (Scheme 1C).…”

“…[12] Cellular retinoic acid-binding protein II (CRABPII) was reengineered to bind all- trans -retinal as a PSB. We were not only able to show wavelength regulation in absorption, as a function of perturbing electrostatic potential across the length of the chromophore, but were also successful in modulating the p K a of the iminium species from 2.4 to 8.1.…”