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Sarah Bernier, Marc-Antoine Millette, Line Cantin, Christian Salesse; Structure and membrane binding of R9AP, a protein involved in phototransduction. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3067.
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Mutations in R9AP (RGS9-1-Anchor-Protein) lead to a visual disease called bradyopsia. R9AP anchors a protein complex to disk membranes of photoreceptor outer segments which inactivate phosphodiesterase and visual phototransduction. This binding of R9AP to photoreceptor membranes was postulated to occur through its C-terminal domain. However, membrane binding and structural data are still lacking for this particular protein.
The present study was thus performed using 1) full-length R9AP, 2) truncated R9AP without its C-terminal domain as well as 3) its C-terminal domain. Infrared and fluorescence spectroscopy as well as circular dichroism were used to gather structural information on R9AP. Langmuir monolayer was used to determine the extent of R9AP membrane binding as well as the importance of its C-terminal domain in this binding.
The truncated protein is much more soluble than the full-length protein and the C-terminal segment is only soluble in particular hydrophobic solvents. Infrared spectroscopy as well as circular dichroism measurements have allowed determining that the truncated protein as well as the C-terminal domain adopt an α-helical structure. Langmuir monolayer measurements revealed that the C-terminal peptide has a clear affinity for most of the phospholipids typical of photoreceptor membranes. In contrast, the truncated protein showed a low affinity for these phospholipids except for unsaturated phosphoserine. Spectrofluorometric measurements using wild-type R9AP and two mutants where each of the two individual tryptophans of R9AP have been replaced by a phenylalanine provided information on the specific environment of these amino acids.
Results have allowed to postulate an important role of the C-terminal domain in the binding and the solubility of R9AP.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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