A dark and constitutively active mutant of the tiger salamander UV pigment

Biochemistry. 2005 Jan 18;44(2):799-804. doi: 10.1021/bi047898f.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Ambystoma / genetics*
  • Ambystoma / metabolism*
  • Amino Acid Substitution / genetics
  • Animals
  • Cattle
  • Darkness*
  • Humans
  • Mutagenesis, Insertional
  • Pigments, Biological / genetics*
  • Pigments, Biological / isolation & purification
  • Pigments, Biological / metabolism*
  • Protein Conformation
  • Retinal Cone Photoreceptor Cells / chemistry
  • Retinal Cone Photoreceptor Cells / metabolism*
  • Retinaldehyde / analogs & derivatives*
  • Retinaldehyde / genetics
  • Retinaldehyde / metabolism
  • Rod Opsins / metabolism
  • Spectrophotometry, Ultraviolet
  • Transducin / metabolism
  • Ultraviolet Rays*

Substances

  • 9-demethylretinal
  • Pigments, Biological
  • Rod Opsins
  • Transducin
  • Retinaldehyde