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.

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.

Membrane interactions of synthetic peptides with antimicrobial potential: effect of electrostatic interactions and amphiphilicity.

Cationic antimicrobial peptides are considered promising candidates to complement currently used antibiotics, which are less effective against increasingly resistant pathogens. To determine the mechanism of action of these peptides, a better understanding of each molecular determinant involved in their membrane interactions is of great importance. In this study, we have focused on the role of electrostatic interactions and amphiphilicity on the membrane interactions since the large majority of natural antimicrobial peptides are cationic. Therefore, cationic and anionic peptides have been prepared based on a model 14-mer peptide. The latter is a synthetic peptide composed of ten leucines and four phenylalanines, which are modified by the addition of the crown ether. Infrared spectroscopy results indicate that the position of substitution is the main determinant involved in the secondary structure adopted by the peptides, and not the charge of the substituted residues. Fluorescence vesicle leakage assays indicate, however, differences between the ability of cationic and anionic peptides to induce calcein release in zwitterionic and anionic lipid vesicles, suggesting an importance of electrostatic interactions and repulsions. Finally, (31)P NMR results indicate that the vesicle morphologies is not significantly affected by the interactions with both cationic and anionic peptides but that their effect on lipid bilayers is mainly determined by their secondary structure. This study therefore indicates that the membrane interactions of model 14-mer peptides are mainly governed by their secondary structure, which depends on the position of substitution, and not the charge of the residues.