Engineering a Coiled Coil Protein as pH Sensor

Abstract

To design our pH sensor, we decided on using an anti-parallel coiled-coil protein. The protein consists of two α-helices connected by a loop, adopting a super-coiled tertiary structure. Designing and engineering of coiled-coil proteins are already parametrized, so length of the helices can be controlled as desired. The approach for our sensor design incorporates the ‘binding coupled to folding’ design-principle with grafting of histidines (H). The partially protonated side chain of histidine has a pKa close to physiological pH and can switch between protonated (general acid) and unprotonated (general base) states. This switching from an aromatic (at higher pH) to positively charged (at lower pH) state, can have a role in (un)stabilizing the folded structure of the protein. We mutated H into the hydrophobic core to manipulate the coiled-coil structure stability, resulting in an intrinsically unstable native state. The protein populates the unfolded state when the concentration of protons is low and as the concentration of protons is increased, the protein ensemble shifts to the folded state. To monitor this transition, we used quenching of fluorescent dye (Atto 655). As the protein folds, photo-induced electron transfer (PET) occurs between the excited state dye and the ground-state of tryptophan (W). Our results show quenching of about 80% of florescence intensity as the pH is lowered from 8-4, also corroborating our circular dichroism studies.