Abstract
Purpose::
In visual phototransduction, it is thought that recoverin, a 190 amino acids calcium myristoyl switch protein, is responsible for the lifetime of light-stimulated phosphodiesterase activity by regulating rhodospin phosphorylation. Recoverin is predominantly acylated with myristic acid, as well as related fatty acids in a minor proportion. Calcium binding to myristoylated recoverin leads to a conformational change, which exposes hydrophobic residues and its mysritoyl. Our objective was to understand at a molecular level how this calcium sensitive protein binds to membranes. In particular, we wished to understand the calcium dependent binding interaction between myristoylated recoverin and model membranes mimicking rod photoreceptor membranes.
Methods::
Photoreceptor membranes have been mimicked by preparing monolayers of phospholipids with different fatty acyl chains (length and unsaturation) and polar headgroups. Recoverin was injected into the subphase underneath these phospholipid monolayers and its binding was monitored by surface pressure measurements. In addition, the function of charged amino acids of recoverin on its membrane binding has been assessed by succinylating its negatively charged amino acids. The number of modified residues has been evaluated by isoelectric focusing and the secondary structure of recoverin by circular dichroism. In addition, fluorescence microscopy has been used to provide information on film morphology and on the macroscopic organization of recoverin-phospholipid films.
Results::
The surface pressure data demonstrate that the affinity of recoverin for phospholipid monolayers increases with fatty acyl chain length and unsaturation. In addition, the use of succinylated recoverin showed that electrostatic interactions are implicated in recoverin binding, as much as hydrophobic interactions. Fluorescence microscopy provided information on the homogeneity of distribution of recoverin in the monolayer.
Conclusions::
Electrostatic and hydrophobic interactions of recoverin are key structural determinants for the interaction between recoverin and rod photoreceptor membranes. This interaction will be further studied by spectroscopic methods, such as polarization modulated infrared reflection spectroscopy (PM-IRRAS).
Keywords: protein structure/function • lipids • linkage analysis