Supplementary Figure 3: Molecular analysis and physiological characterization of Jaws variants.
From: Noninvasive optical inhibition with a red-shifted microbial rhodopsin
(a) Sequence alignment of Halo57, the H. salinarum strain shark cruxhalorhodopsin, with the N. pharaonis halorhodopsin, demonstrating < 60% sequence homology; blue residues denote sequence divergence, and grey residues, conservation. (b) Schematic of Halo57, Jaws, and Jaws-ER2 proteins. Black denotes the Halo57 protein scaffold, red indicates the K200R and W214F point mutations, green indicates the C terminal GFP fusion, and KGC and ER2 respectively refer to endoplasmic reticulum forward transport and Golgi export sequences from the potassium channel Kir2.1. (c-d) The K200R W214F mutation boosts Halo57 photocurrents (n = 3 cells for wildtype, n = 6 cells for K200R W214F mutant), but the homologous K215R W229F mutations cause no effect in eNpHR3.0 at 5 mW/mm2 (c) or across a range of 632 nm light powers (d; n = 3 cells for both wildtype and mutant eNpHR3.0). (e) Current-voltage relationship for light-activated Jaws photocurrents (n = 5 cells; 632 nm, 5 mW/mm2). Error bars are smaller than the symbols plotted. (f) Jaws photocurrent is dependent on [Cl-] in the extracellular bath solution (n = 3-5 cells; 632 nm, 5 mW/mm2). All measurements were taken in HEK293FT cells; values are mean ± standard error.
