Characterization of neurocalcin delta membrane binding by biophysical methods.

Neurocalcin delta (NCALD) is a member of the neuronal calcium sensors protein family. In the retina, NCALD is expressed by ganglion and amacrine cells. NCALD is composed of 4 EF-hand motifs but only 3 of them may bind calcium. The binding of calcium induces a conformational change of the protein which leads to the extrusion of its N-terminal myristoyl group as well as some hydrophilic residues. This mechanism, named calcium-myristoyl switch, is presumably involved in its membrane binding. The parameters responsible for the interaction of NCALD with membranes are only partially known. The purpose of this study was thus to gather more information on the membrane binding behavior of NCALD using lipid monolayers, including the influence of the lipid composition, the calcium and the myristoyl group. NCALD was injected underneath different lipid monolayers and this model membrane allowed the determination of the binding parameters as maximum insertion pressure (MIP) and synergy. The values of MIP are larger when monolayers were composed of a saturated phospholipid with phosphoethanolamine polar head. This trend is confirmed by polarization modulation infrared reflection absorption spectroscopy measurements. Moreover, the observations by fluorescence microscopy show that NCALD preferentially interacts with phospholipids which are in the liquid-condensed physical state, as found in membrane microdomains. This observation could explain the changes of NCALD expression level in the brains of patients suffering from Alzheimer's disease because of the alteration of lipid composition in microdomains structures.

Lipid Selectivity, Orientation, and Extent of Membrane Binding of Nonacylated RP2.

Retinitis pigmentosa 2 (RP2) is an ubiquitary protein of 350 residues. The N-terminus of RP2 contains putative sites of myristoylation and palmitoylation. The dually acylated protein is predominantly localized to the plasma membrane. However, clinically occurring substitution mutations of RP2 in photoreceptors lead to the expression of a nonacylated protein, which was shown to be misrouted to intracellular organelles using different cell lines. However, the parameters responsible for the modulation of the membrane binding of nonacylated RP2 (naRP2) are still largely unknown. The maximal insertion pressure of naRP2 has thus been determined after its injection into the subphase underneath monolayers of phospholipids, which are typical of photoreceptor membranes. These data demonstrated that naRP2 shows a preferential binding to saturated phospholipid monolayers. Moreover, polarization modulation infrared reflection absorption spectroscopy has allowed comparison of the secondary structure of this protein in solution and upon binding to phospholipid monolayers. In addition, simulations of these spectra have allowed to determine that the ß-helix of naRP2 has an orientation of 60° with respect to the normal, which remains unchanged regardless of the type of phospholipid. Finally, ellipsometric measurements of naRP2 demonstrated that its particular affinity for saturated phospholipids can be explained by its larger extent of insertion in this phospholipid monolayer compared to that in polyunsaturated phospholipid monolayers.