Role of the Myristoyl Moiety and Effect of Ions in the Membrane Binding of Recoverin by PM–IRRAS and Surface Pressure Measurements

Abstract

Abstract: : Purpose: Myristoyl switch is a feature of several peripheral membrane proteins. This unique molecular property is best illustrated by the ‘calcium–myristoyl switch’ of recoverin (Rv) which is a calcium–binding protein present in retinal rod cells of vertebrates. Rv contains an amino–terminal myristoyl group (Myr) and acts as a calcium sensor by regulating the rod cell response to the changes in intracellular calcium. Indeed, the binding of two calcium ions to Rv induces the extrusion of its Myr which increases its affinity for membranes. However, in the calcium–free form, the Myr is sequestered in a hydrophobic cleft of Rv. Little is known about the parameters responsible for the modulation of the membrane binding of Rv. Consequently, this study was performed to determine the involvement of the charged amino acids of Rv as well as its Myr in its membrane binding. Methods: Polarization–modulated infrared reflection absorption spectroscopy (PM–IRRAS) combines Fourier transform mid–IR reflection spectroscopy with rapid polarization modulation of the incident beam. This spectroscopic method is very sensitive to characterize thin films spread at the air/water interface. We have thus used PM–IRRAS and Surface Pressure Measurements (SPM) to measure Rv adsorption onto phospholipid monolayers at the air–water interface and to probe protein structure in presence of different ions and phospholipids. Results: PM–IRRAS spectra indicate that Rv contains a major proportion of α–helix and that its folding is preserved at the air/water interface. SPM of the adsorption of myristoylated and unmyristoylated Rv shows clearly that both the myristoyl group and hydrophobic residues contribute to Rv membrane binding. Conclusions: Rv preferentially binds certain phospholipids and ions other than calcium can increase its affinity for membranes. These results allow a better understanding of the parameters responsible for the membrane binding of Rv and also for peripheral proteins in general.

Keywords: protein structure/function • calcium • lipids