Calcium-Induced Formation of Subdomains in Phosphatidylethanolamine−Phosphatidylglycerol Bilayers: A Combined DSC, 31P NMR, and AFM Study

Abstract

We study the effect of Ca²⁺ on the lateral segregation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). Supported lipid bilayers (SLBs) were observed by atomic force microscopy (AFM). Since SLBs are formed from liposomes of POPE:POPG, we examined the effect of calcium on these suspensions by differential scanning calorimetry (DSC) and ³¹P nuclear magnetic resonance spectroscopy (³¹P NMR). AFM images revealed the existence of two separated phases, the higher showing a region with protruding subdomains. Force spectroscopy (FS) was applied to clarify the nature of each phase. The values of breakthrough force (F_y), adhesion force (F_adh), and height extracted from the force curves were assigned to the corresponding gel (L_β) and fluid (L_α) phase. The endotherms obtained by DSC suggest that, in the presence of Ca²⁺, phase separation already exists in the suspensions of POPE:POPG used to form SLBs. Due to the temperature changes applied during preparation of SLBs a ³¹P NMR study was performed to assess the lamellar nature of the samples before spreading them onto mica. With in situ AFM experiments we showed that the binding of Ca²⁺ to POPG-enriched domains only induces the formation of subdomains in the L_β phase.