Novel approaches to probe the binding of recoverin to membranes

• Published in: European biophysics journal Vol. 47; no. 6; pp. 679 - 691
• Main Authors: Potvin-Fournier, Kim, Valois-Paillard, Geneviève, Gagnon, Marie-Claude, Lefèvre, Thierry, Audet, Pierre, Cantin, Line, Paquin, Jean-François, Salesse, Christian, Auger, Michèle
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01-09-2018; Springer Nature B.V
• Subjects: Binding; Biochemistry; Biological and Medical Physics; Biomedical and Life Sciences; Biophysics; Calcium; Calcium ions; Cell Biology; Fluorine; Hydrophobicity; Immobilization; Infrared spectroscopy; Lateral diffusion; Lateral stability; Lecithin; Life Sciences; Lipids; Membrane Biology; Membranes; Nanotechnology; Neurobiology; NMR; Nuclear magnetic resonance; Original Article; Phosphatidylcholine; Phototransduction; Recoverin; Rhodopsin kinase; Rod outer segment membranes; Thermal stability; Phosphatidylcholine; Lateral diffusion of lipids; Infrared spectroscopy; Solid-state nuclear magnetic resonance spectroscopy; Fluorine; Multilamellar vesicles
• ISSN: 0175-7571; 1432-1017
• DOI: 10.1007/s00249-018-1304-4

Recoverin is a protein involved in the phototransduction cascade by regulating the activity of rhodopsin kinase through a calcium-dependent binding process at the surface of rod outer segment disk membranes. We have investigated the interaction of recoverin with zwitterionic phosphatidylcholine bilayers, the major lipid component of the rod outer segment disk membranes, using both
31
P and
19
F solid-state nuclear magnetic resonance (NMR) and infrared spectroscopy. In particular, several novel approaches have been used, such as the centerband-only detection of exchange (CODEX) technique to investigate lipid lateral diffusion and
19
F NMR to probe the environment of the recoverin myristoyl group. The results reveal that the lipid bilayer organization is not disturbed by recoverin. Non-myristoylated recoverin induces a small increase in lipid hydration that appears to be correlated with an increased lipid lateral diffusion. The thermal stability of recoverin remains similar in the absence or presence of lipids and Ca
2+
. Fluorine atoms have been strategically introduced at positions 4 or 12 on the myristoyl moiety of recoverin to, respectively, probe its behavior in the interfacial and more hydrophobic regions of the membrane.
19
F NMR results allow the observation of the calcium–myristoyl switch, the myristoyl group experiencing two different environments in the absence of Ca
2+
and the immobilization of the recoverin myristoyl moiety in phosphatidylcholine membranes in the presence of Ca
2+
.