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 die basis of the data, the protein predominantly adopts a-helical structures (∼50-55%) with turns, unordered staictures, and β-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 β-sheets. The nonmyristoylated protein (Rec-nMyr) exhibits a lower temperature threshold of aggregation and a higher intermolecular β-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, tiie presence of the myristoyl chain having no effect on the thermal stability of recoverin under these conditions. A decrease in the thermal stability oi 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. [ABSTRACT FROM AUTHOR]