Novel approaches to probe the binding of recoverin to membranes.

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 31P and 19F 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 19F 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 Ca2+. 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. 19F NMR results allow the observation of the calcium-myristoyl switch, the myristoyl group experiencing two different environments in the absence of Ca2+ and the immobilization of the recoverin myristoyl moiety in phosphatidylcholine membranes in the presence of Ca2+.

Membrane fluidity is a driving force for recoverin myristoyl immobilization in zwitterionic lipids.

Recoverin is the only protein for which the phenomenon of calcium-myristoyl switch has been demonstrated without ambiguity. It is located in rod disk membranes where the highest content in polyunsaturated lipid acyl chains can be found. However, although essential to better understand the inactivation of the phototransduction process, the role of membrane fluidity on recoverin recruitment is unclear. We have therefore investigated the immobilization of the recoverin myristoyl moiety in the presence of phosphocholine bilayers using 2H solid-state NMR spectroscopy. Several lipids with different acyl chains were selected to investigate model membranes characterized by different fluidity. Immobilization of the recoverin myristoyl moiety was successfully observed but only in the presence of calcium and in specific lipid disordered states, showing that an optimal fluidity is required for recoverin immobilization.

Discriminating Lipid- from Protein-Calcium Binding To Understand the Interaction between Recoverin and Phosphatidylglycerol Model Membranes.

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. Understanding how calcium modulates these interactions and how it interacts with anionic lipid membranes is necessary to gain insights into the function of recoverin. In this work, infrared spectroscopy allowed us to show that the availability of calcium to recoverin is modulated by the presence of complexes involving phosphatidylglycerol (PG), which in turn regulates its interactions with this negatively charged lipid. Calcium can indeed be sequestered into strongly bound complexes with PG and is thus sparingly available to recoverin. The thermal stability of recoverin then decreases, which results in weakened interactions with PG. By contrast, when calcium is fully available to recoverin, the protein is thermally stable, indicating that it binds two calcium ions, which results in favorable interactions with negatively charged lipids. Consequently, the protein induces an increase in the chain-melting phase transition temperature of PG, which is indicative of an enhanced lipid chain packing resulting from the peripheral location of the protein. The secondary structure of recoverin is not affected by its interactions with anionic membrane lipids. Similar results have been obtained with saturated and unsaturated anionic lipids. This work shows that the recruitment of recoverin at the surface of anionic lipid membranes is dependent on the availability of calcium.

The thermal stability of recoverin depends on calcium binding and its myristoyl moiety as revealed by infrared spectroscopy.

To evaluate the structural stability of recoverin, a member of the neuronal calcium sensor family, the effect of temperature, myristoylation, and calcium:protein molar ratio on its secondary structure has been studied by transmission infrared spectroscopy. On the basis of the data, the protein predominantly adopts α-helical structures (∼50-55%) with turns, unordered structures, and ß-sheets at 25 °C. The data show no significant impact of the presence of calcium and myristoylation on secondary structure. It is found that, in the absence of calcium, recoverin denatures and self-aggregates while being heated, with the formation of intermolecular antiparallel ß-sheets. The nonmyristoylated protein (Rec-nMyr) exhibits a lower temperature threshold of aggregation and a higher intermolecular ß-sheet content at 65 °C than the myristoylated protein (Rec-Myr). The former thus appears to be less thermally stable than the latter. In the presence of excess calcium ions (calcium:protein ratio of 10), the protein is thermally stable up to 65 °C with no significant conformational change, the presence of the myristoyl chain having no effect on the thermal stability of recoverin under these conditions. A decrease in the thermal stability of recoverin is observed as the calcium:protein molar ratio decreases, with Rec-nMyr being less stable than Rec-Myr. The data overall suggest that a minimal number of coordinated calcium ions is necessary to fully stabilize the structure of recoverin and that, when bound to the membrane, i.e., when the myristoyl chain protrudes from the interior pocket, recoverin should be more stable than in a Ca-free solution, i.e., when the myristoyl chain is sequestered in the interior.