Regulation of photoactivation in vertebrate short wavelength visual pigments: protonation of the retinylidene Schiff base and a counterion switch.
Xenopus violet cone opsin (VCOP) and its counterion variant (VCOP-D108A) are expressed in mammalian COS1 cells and regenerated with 11-cis-retinal. The phototransduction process in VCOP-D108A is investigated via cryogenic electronic spectroscopy, homology modeling, molecular dynamics, and molecular orbital theory. The VCOP-D108A variant is a UV-like pigment that displays less efficient photoactivation than the mouse short wavelength sensitive visual pigment (MUV) and photobleaching properties that are significantly different. Theoretical calculations trace the difference to the protonation state of the nearby glutamic acid residue E176, which is the homology equivalent of E181 in rhodopsin. We find that E176 is negatively charged in MUV but neutral (protonated) in VCOP-D108A. In the dark state, VCOP-D108A has an unprotonated Schiff base (SB) chromophore (lambdamax = 357 nm). Photolysis of VCOP-D108A at 70 K generates a bathochromic photostationary state (lambdamax = 380 nm). We identify two lumi intermediates, wherein the transitions from batho to the lumi intermediates are temperature- and pH-dependent. The batho intermediate decays to a more red-shifted intermediate called lumi I. The SB becomes protonated during the lumi I to lumi II transition. Decay of lumi II forms meta I, followed by the formation of meta II. We conclude that even in the absence of a primary counterion in VCOP-D108A, the SB becomes protonated during the photoactivation cascade. We examine the relevance of this observation to the counterion switch mechanism of visual pigment activation.
Ramos
Lavoisier S
LS
Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, USA.
Chen
Min-Hsuan
MH
Knox
Barry E
BE
Birge
Robert R
RR
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011522:Protons; D017949:Retinal Cone Photoreceptor Cells / Q000737:chemistry / Q000378:metabolism*; D012168:Retinal Pigments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012176:Retinoids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017299:Rod Opsins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012545:Schiff Bases / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D040681:Structural Homology, Protein; D014785:Vision, Ocular / Q000235:genetics; D014982:Xenopus laevis
D011522:Protons; D012168:Retinal Pigments; D012176:Retinoids; D017299:Rod Opsins; D012545:Schiff Bases; C121835:retinylidene chromophore; C095853:short-wavelength opsin; D001224:Aspartic Acid