Abstract
Naturally occurring point mutations in the opsin gene cause the retinal diseases retinitis pigmentosa and congenital night blindness. Although these diseases involve similar mutations in very close locations in rhodopsin, their progression is very different, with retinitis pigmentosa being severe and causing retinal degeneration. We report on the expression and characterization of the recently found T94I mutation associated with congenital night blindness, in the second transmembrane helix or rhodopsin, and mutations at the same site. T94I mutant rhodopsin folded properly and was able to bind 11-cis-retinal to form chromophore, but it showed a blue-shifted visible band at 478 nm and reduced molar extinction coefficient. Furthermore, T94I showed dramatically reduced thermal stability, extremely long lived metarhodopsin II intermediate, and highly increased reactivity toward hydroxylamine in the dark, when compared with wild type rhodopsin. The results are consistent with the location of Thr-94 in close proximity to Glu-113 counterion in the vicinity of the Schiff base linkage and suggest a role for this residue in maintaining the correct dark inactive conformation of the receptor. The reported results, together with previously published data on the other two known congenital night blindness mutants, suggest that the molecular mechanism underlying this disease may not be structural misfolding, as proposed for retinitis pigmentosa mutants, but abnormal functioning of the receptor by decreased thermal stability and/or constitutive activity.
Highlights
Occurring mutations in rhodopsin are associated with retinal disease
We report on the expression and characterization of the recently found T94I mutation associated with congenital night blindness, in the second transmembrane helix or rhodopsin, and mutations at the same site
Similar elution profiles were obtained for the wild type and the T94I and T94D mutants (Fig. 2, A–C) with most of the protein eluting with pH 6 buffer, with no salt, in the third fraction, and only a very small amount of non-retinal binding fraction eluting with added salt
Summary
Materials—11-cis-Retinal was purified from illuminated all-transretinal using a modified version of a protocol described previously [15]. The buffers used are as follows: buffer A, 1.8 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, pH 7.2; buffer B, buffer A plus 5 mM ATP, 5 mM MgCl2, 0.1 mM phenylmethylsulfonyl fluoride, and 1% (w/v) DM; buffer C, buffer A plus 0.05% DM; buffer D, 2 mM NaH2PO4, pH 6.0, 0.05% DM; buffer E, buffer D plus 150 mM NaCl. Cloning, Expression, and Purification of Thr-94 Mutants—Construction of the mutant opsin genes at position 94 was carried out by replacement of the BglII-NcoI restriction fragment in the synthetic opsin gene in the pMT4 vector [16]. The column was subsequently washed with 10 ml of buffer C, followed by 10-ml wash with buffer D at the same rate, and eluted in buffer D containing 100 M of CЈ1–9 peptide, at a rate of 60 l/min. 2 g of protein in 200 l of buffer D were allowed to equilibrate at 20 °C for 30 min, bleached for 30 s, and the fluorescence increase measured. Spectra were normalized and fitted to single exponential functions using SigmaPlot to derive the t1⁄2 values
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