April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Regulation of Photosensitivity in a Cone Visual Pigment
Author Affiliations & Notes
  • B. E. Knox
    Biochem & Molecular Bio and Ophthal, SUNY Upstate Medical Univ, Syracuse, New York
  • C. M. Kuemmel
    Biochem & Molecular Bio and Ophthal, SUNY Upstate Medical Univ, Syracuse, New York
  • Footnotes
    Commercial Relationships  B.E. Knox, None; C.M. Kuemmel, None.
  • Footnotes
    Support  National Eye Institute/NIH (EY011256, EY012975); Unrestricted grant from Research to Prevent Blindness.
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 1100. doi:
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      B. E. Knox, C. M. Kuemmel; Regulation of Photosensitivity in a Cone Visual Pigment. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1100.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Rods and cones function in different light intensity realms, yet all visual pigments (VP) have similar photosensitivities (Makino et al., 1991 J.Physiol. 442, 761) implying conserved mechanisms of efficient VP photoactivation. Photosensitivity is a product of the intense absorptivity of the retinal chromophore and high quantum yield for 11-cis to all-trans isomerization. Vertebrate VP employ either A1 or A2-retinal with very similar extinction coefficients, so the quantum yield must also be similar. How do VP achieve similar quantum yields? We have studied the molecular basis for the quantum efficiency of the photoactivation of a cone visual pigment.

Methods: : Interhelical residues between TM5-TM6 in a Xenopus short-wavelength VP (VCOP) were targeted for mutagenesis. Site-specific mutagenesis, VCOP expression in COS1 cells and purification in delipidated form with dodecyl maltoside was performed according to standard protocols. UV-visible absorption spectra were recorded at various temperatures (Kusnetzow et al., 2004, PNAS 101, 941). Retinal binding to VP was measured using a time-based fluorescence quenching assay (Farrens & Khorana, 1995, JBC 270, 5073).

Results: : In a short wavelength cone VP, we found a conserved aromatic residue at an interhelical position that was sensitive to substitution. Only smaller or hydrophobic side chains were tolerated, producing proteins that bound 11-cis retinal with efficiency and molar absorptivity (extinction coefficient) similar to wild-type VCOP. One mutant required at least three times as many photons for bleaching as wild-type. A reduced photosensitivity of the mutant versus wild-type was confirmed in light-dependent all-trans retinal release assays. Other mutants at this position exhibited variable photoactivation behaviors. By contrast, mutation of other non-conserved aromatic residue on the same interhelical interface did not alter photosensitivity.

Conclusions: : A conserved aromatic residue in the TM5-6 interhelical interface modulates protein-chromophore interactions, as measured by binding of 11-cis retinal to mutant VPs. This interface is important for high photosensitivity and potentially for all-trans retinal release. Since mutant and wild-type VPs had similar extinction coefficients, we conclude that TM5-6 interface is an important determinant of the high quantum yield of VP.

Keywords: color pigments and opsins • protein structure/function • photoreceptors 

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