Stability of Alternative Bacteriorhodopsin Folds

Abstract

Bacteriorhodopsin is a light-activated proton pump found in archaea and some single-celled eukaryotes (Findlay and Pappin, 1986; Sharma et al., 2006; Spudich et al., 2000). This protein adopts the 7TM fold found used by all type I and type II rhodopsins. In previous work, we used bacteriorhodopsin from Haloterrigena turkmenica to demonstrate that substantially altered protein folds exhibit light-activated proton pumping (Kamo et al., 2006; Mackin et al., 2014). In this work, we further characterized these novel folds by assessing the stability of our mutants. We used SDS denaturation to calculate the change in unfolding free energy relative to the wildtype (Cao et al., 2012). We also determine the extinction coefficient for each mutant. These results demonstrate that even dramatic structural rearrangements do not critically destabilize the protein, although the extinction coefficient does vary independently of the stability and the proton-pumping activity. Interestingly, the position of the A helix in the protein sequence has the largest effect on the stability of the mutant; those mutants where A is not located at a terminus are destabilized compared to the wild-type.