Novel approaches to probe the binding of recoverin to membranes

Abstract

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 P andF 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 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. 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. F NMR results allow the observation of the calcium-myristoyl switch, the myristoyl group experiencing two different environments in the absence of Ca and the immobilization of the recoverin myristoyl moiety in phosphatidylcholine membranes in the presence of Ca.

Binding of a Myristoylated Protein to the Lipid Membrane Influenced by Interactions with the Polar Head Group Region

“…The binding of recoverin to ROS membranes is energetically mainly driven by insertion of the exposed myristoyl chain into a fluid acyl chain region of the bilayer. ,,− Recoverin has an enhanced affinity to membrane fragments, which are rich in cholesterol . Moreover, other factors such as charge–charge and/or charge–dipole interactions between the polar head groups and positive charge at the polybasic cluster of side chains at the N-terminus of recoverin contribute to the lipid–protein interactions. ,,− Since years, PCs are used as models of cell membranes in studies of lipid–recoverin interaction. ,,, The liquid state of the acyl chains in 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) and the presence of cholesterol in the model lipid bilayer provide good environment for the study of lipid–recoverin interactions.…”

“…4,15,18−25 Since years, PCs are used as models of cell membranes in studies of lipid−recoverin interaction. 2,4,25,26 The liquid state of the acyl chains in 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC) and the presence of cholesterol in the model lipid bilayer provide good environment for the study of lipid−recoverin interactions. NMR and molecular dynamic studies of binding of myristoylated recoverin to the lipid membrane indicate that at the N-terminus the amino acids K5, K11, K37, R43, and K84 make a close contact with the polar head group region of the bilayer.…”

The Binding Properties and Physiological Functions of Recoverin

“…Little is known about how the insertion of the myristoyl moiety affects the lipid membrane. Although the insertion does not seem to disturb membrane integrity (Potvin-Fournier et al, 2018), it greatly impacts the electrochemical properties of the bilayer (Brand and Koch, 2018). Both the surface charge density and the surface pressure of membranes are reduced upon Rcv binding, supporting the importance of electrostatic interactions.…”