Vertebrate ultraviolet visual pigments: Protonation of the retinylidene Schiff base and a counterion switch during photoactivation

Abstract

For visual pigments, a covalent bond between the ligand (11-cisretinal) and receptor (opsin) is crucial to spectral tuning and photoactivation. All photoreceptors have retinal bound via a Schiff base (SB) linkage, but only UV-sensitive cone pigments have this moiety unprotonated in the dark. We investigated the dynamics of mouse UV (MUV) photoactivation, focusing on SB protonation and the functional role of a highly conserved acidic residue (E108) in the third transmembrane helix. On illumination, wild-type MUV undergoes a series of conformational changes, batho 3 lumi 3 meta I, finally forming the active intermediate meta II. During the dark reactions, the SB becomes protonated transiently. In contrast, the MUV-E108Q mutant formed significantly less batho that did not decay through a protonated lumi. Rather, a transition to meta I occurred above Ϸ240 K, with a remarkable red shift ( max Ϸ 520 nm) accompanying SB protonation. The MUV-E108Q meta I 3 meta II transition appeared normal but the MUV-E108Q meta II decay to opsin and free retinal was dramatically delayed, resulting in increased transducin activation. These results suggest that there are two proton donors during the activation of UV pigments, the primary counterion E108 necessary for protonation of the SB during lumi formation and a second one necessary for protonation of meta I. Inactivation of meta II in SWS1 cone pigments is regulated by the primary counterion. Computational studies suggest that UV pigments adopt a switch to a more distant counterion, E176, during the lumi to meta I transition. The findings with MUV are in close analogy to rhodopsin and provides further support for the importance of the counterion switch in the photoactivation of both rod and cone visual pigments.V isual pigments are seven transmembrane ␣-helical proteins that initiate the light transduction pathway in retinal photoreceptors. Whereas other G protein-coupled receptors interact with their ligands noncovalently, the visual pigments consist of 11-cis-retinal covalently attached to the apoprotein via a Schiff base (SB) linkage to a conserved lysine in transmembrane helix 7 (TM7) (Fig. 1). After absorption of light, the retinal chromophore isomerizes to the all-trans conformation and triggers a series of conformational changes that lead to the formation of the active state, R* or meta II. All-trans-retinal is eventually released from the vertebrate apoprotein, and the visual pigment can be regenerated with 11-cis-retinal.In dark-adapted rhodopsin, the SB pK a is extraordinarily high resulting in a protonated SB buried within the chromophore binding site (1). The protonation is important to prevent the spontaneous hydrolysis of the SB and contributes to the max (2, 3). The binding site of rhodopsin is neutral (4), and E113 serves as a counterion to the protonated SB (5-8). The stability of the salt bridge is enhanced by a single water molecule that interacts with the side chains of E113 and indirectly through a second water molecule that interacts with adjacent residues (9,...

Signaling Properties of a Short-Wave Cone Visual Pigment and Its Role in Phototransduction

“…We found that it decays ϳ40 times faster than rhodopsin meta II, with a time constant of ca. 1.3 s. This value, derived in situ at 37°C, should be more precise than the previous in vitro biochemical estimate of Ͻ30 s at 22°C for mouse S-pigment (Kusnetzow et al, 2004) or 12 min at Ϫ10°C for chicken S-pigment (Imai et al, 1997). Although the meta II lifetime normally does not dominate the decay of the cone light response, the amount of cone pigment activated by intense light may exceed the cell capacity for phosphorylation such that the rapid decay of meta II may become important for response decline.…”

The Retinal Conformation and its Environment in Rhodopsin in Light of a New 2.2 Å Crystal Structure

“…41 The network may form the basis for the counterion switch from Glu113 to Glu181 during formation of the meta I state of rhodopsin. [42][43][44] In the dark state it stabilizes the peculiar charge distribution of the chromophore at the binding site. Our calculations 39,40 have shown that a major contributing factor for keeping the Schiff base nitrogen atom protonated is the involvement of Glu113 in hydrogen bonding, which reduces its basicity.…”

How a small change in retinal leads to G‐protein activation: Initial events suggested by molecular dynamics calculations

“…11,32,[54][55][56][57][58][59][60][61][62][63][64][65] This would involve a change in protonation states during the photocycle 33 and experimental work has confirmed that Glu 113 is the dark-state counterion and been suggestive, but not conclusive, that Glu 181 is active during the light-activated stages. 66,67 The simulation results are intriguing in suggesting that this counterion switch mechanism could be present as part of the relaxation mechanism of the protein to the retinal conformational change.…”

“…We should emphasize that in our calculations there is no change in the charge state of Glu 181 during or after the forced isomerization. 57,68 Thus, the driving force for the change is not wholly from charge transfer during the photocycle, but could also be related to the coupled relaxation of the full system from the initial events of light activation. In this regard, quantum calculations about the effect of the counterion switch are also suggestive of the types of changes occurring with a counterion switch that could underlie function.…”