Fig. 4.

Osmotic pressure induces large shifts of pH-dependent activation of rhodopsin. (A) Influences of pH on rhodopsin are described by phenomenological Henderson-Hasselbalch equation involving two pK_A values and an alkaline endpoint. The states are distinguished by having a protonated or deprotonated Schiff base (PSB or SB, indicated by a subscript), while a superscript indicates the charge relative to MI. The lower pK_A (designated pK_A,SB) reflects pH-dependent protonation of the retinal Schiff base (SB), which lowers the apparent MII fraction detected by UV-visible spectroscopy. The higher pK_A value (designated pK_A,Glu) reflects protonation of Glu¹³⁴ in the E(D)RY motif to stabilize the fully active MII conformation. The alkaline endpoint at higher pH corresponds to MII substates that persist at higher temperatures even when Glu¹³⁴ is fully deprotonated. (B) Applied osmotic stress stemming from large osmolytes (50% wt/wt at T = 15 °C) back shifts the rhodopsin activation titration curve from pK_A = 7.4 to 5.2. At 30% wt/wt PEG 200 (T = 15 °C) the titration curve is maximally forward shifted to a pK_A of 8.2 favoring the active MII state. Also observed is an osmolyte effect on the alkaline endpoint: small osmolytes stabilize the open MII conformation even when Glu¹³⁴ is fully deprotonated, increasing the alkaline endpoint, whereas dehydrating large osmolytes decrease the deprotonated MII population and thus the alkaline endpoint.