Influence of the Membrane Composition of Retinal Photoreceptors on the Reversible Binding of a Neuronal Calcium Sensor Protein (NCS), Recoverin : A Solid-State NMR and FTIR Study

Abstract

NCS proteins consist of 4 domains called EF-Hand, which are 2 α-helices joined by a loop with a highly conserved sequence. Binding of calcium ions by this loop induces important conformational changes in the protein. Most of the NCS also have a N-terminal sequence recognized by N-myristoyl transferase which is responsible for the acylation of the proteins. At low calcium concentration, the myristoyl group is sequestered into a hydrophobic cavity. The binding of 1 to 4 Ca2+ leads to the extrusion of the myristoyl (known as the calcium myristoyl switch) and the exposure of many hydrophobic residues allowing the protein to go from a cytosolic form to a membrane bound form. This property has an important biological function in the visual phototransduction cascade. In fact, the absorption of a photon by the visual pigment rhodopsin leads to an important decrease of calcium level in photoreceptors and recoverin, one of the NCS family, plays a key role in the recovery phase of visual excitation by inhibiting rhodopsin kinase at high calcium level. An interesting fact is that the membrane composition of the photoreceptor rod outer segments is known to be very different from that of other membranes, with more than 60% of the lipids being polyinsaturated. We have determined in the present study how the membrane composition affects the reversible membrane binding of recoverin. More specifically, 31P solid-state NMR and 2H solid-state NMR have been used to get information respectively about the lipid polar head groups and the recoverin myristoyl group. FTIR was also used to study the structure of recoverin (amide I’ band) and the lipid acyl chains (CH2 symmetric stretch band).