Preferential Binding and Orientation of Recoverin to Phospholipid Monolayers

Abstract

Recoverin is a 201 amino acids calcium-myristoyl switch protein that is responsible for the regulation of the phosphorylation of the visual pigment rhodopsin. Calcium binding to myristoylated recoverin leads to a conformational change, which exposes its hydrophobic residues and its myristoyl moiety. We have previously demonstrated that the myristoyl group highly accelerates the membrane binding of recoverin in the presence of calcium. However, it is still unknown whether recoverin shows preferential membrane binding towards highly polyunsaturated phospholipids such as those found in photoreceptor membranes. In this study, we performed monolayer measurements to analyze the affinity of recoverin for different phospholipids that are representative of these membranes. We concluded that the affinity of recoverin increases with fatty acyl chain length and unsaturation of the phospholipids. In addition, we observed a preferential binding of recoverin for didocosahexaenoyl phosphatidylethanolamine with a maximum insertion pressure of 30 mN/m, which is in the range of the lateral pressure postulated for biological membranes. Moreover, results show that the size and the charge of the polar head group of phospholipids are also implicated in recoverin adsorption in monolayers. Furthermore, polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) has been used to determine the secondary structure and orientation of recoverin in monolayers. PM-IRRA spectra indicated the prevalence of α-helices in the secondary structure of recoverin, which is consistent with its known structure. In contrast, non-myristoylated recoverin is quickly denatured after its adsorption in monolayers. Finally, the amide I/amide II ratio allowed to determine that the α-helices of myristoylated recoverin are oriented perpendicular to the plane of the monolayer.