A dark and constitutively active mutant of the tiger salamander UV pigment.
A triple mutant (F86L/T93P/S118T; bovine rhodopsin numbering) of the tiger salamander UV cone pigment appears to be trapped in an open conformation that is metarhodopsin-II-like. The pigment is able to activate transducin in the dark, and the ligand-free apoprotein is also able to activate transducin constitutively. The pigment permits protons and chloride ions from solution access to the active site as it displays a pH- and NaCl-dependent absorption spectrum not observed with the wild-type pigment. However, the wild-type properties of light-dependent activity and a pH-independent absorption spectrum are recovered upon reconstitution of the triple mutant with 11-cis-9-demethyl retinal. These results suggest that binding the native chromophore cannot deactivate the protein because of steric interactions between the protein, possibly residue 118, and the 9-methyl group of the chromophore. Furthermore, the absorption spectrum of the 9-demethyl retinal regenerated pigment exhibits a band broader and with lower extinction at the absorption maximum than either the human blue or salamander UV wild-type pigments generated with the same retinal analogue. The broad spectrum appears to be comprised of two or more species and can be well-fit by a sum of scaled spectra of the two wild-type pigments. Binding the chromophore appears to trap the pigment in two or more conformations. The triple mutant reported here represents the first example of a dark-active cone pigment and constitutively active cone opsin.
Kono
Masahiro
M
Department of Ophthalmology, Medical University of South Carolina, Charleston, South Carolina 29425, USA. konom@musc.edu
Crouch
Rosalie K
RK
Oprian
Daniel D
DD
D000557:Ambystoma / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002417:Cattle; D003624:Darkness; D006801:Humans; D016254:Mutagenesis, Insertional; D010860:Pigments, Biological / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D017949:Retinal Cone Photoreceptor Cells / Q000737:chemistry / Q000378:metabolism*; D012172:Retinaldehyde / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D017299:Rod Opsins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D015293:Transducin / Q000378:metabolism; D014466:Ultraviolet Rays
C110176:9-demethylretinal; D010860:Pigments, Biological; D017299:Rod Opsins; D015293:Transducin; D012172:Retinaldehyde