Interaction of cytochrome c with cubic monoolein mesophases at limited hydration conditions: The effects of concentration, temperature and pressure

Abstract

Incorporation of the protein cytochrome c (cyt c) into hydrated cubic mesophases of monoolein (MO) was investigated within a wide range of concentrations, temperatures and pressures by small-angle X-ray scattering (SAXS) and Fourier-transform infrared (FT-IR) spectroscopy. The SAXS data allow the determination of the topology of the mesophase and phase behavior of the lipid system. Further information on the lipid conformational states and hydrational properties were obtained using FT-IR spectroscopy. The effect of the lipid matrix on the structure and stability of the embedded cyt c was also studied by FT-IR spectroscopy by monitoring changes in secondary structural properties of the protein by analysis of the FT-IR amide I′ band. Incorporation of cyt c into the cubic phase of MO has significant effects on the structure and pressure stability of the system and the lipid bilayer and protein molecules undergo interrelated changes in their structural properties, even though the lipids are uncharged. Entrapping of low concentrations of cyt c (< 0.2 wt%) leads to changes only in the phase transition temperatures and pressures. In contrast, higher protein concentrations have a drastic effect on the molecular properties of the lipid system. Lipid–protein interactions lead to an increase in interfacial curvature which promotes the formation of a new, possibly partially micellar, cubic phase of crystallographic space group P4₃32 (Q²¹²). It is very likely that the protein molecules are accommodated in the aqueous interior of inverted micellar aggregates in this structure. The IR data reveal that the new lipid aggregate structure is accompanied by an increase of carbonyl headgroup hydration and a decrease of lipid chain order. As a consequence of attractive protein–headgroup interactions, the pressure stability of this phase increases with increasing the protein content. Above ca. 4 kbar, ordered lamellar phases are induced which have the smallest partial molar volume. The unfolding temperature of cyt c is reduced by ca. 4 °C by incorporation of the protein into the lipid mesophases, as compared to the protein dissolved in water. Contrary to what would be expected for a pure confinement effect, which would lead to a significant increase of the unfolding temperature of the protein, our data clearly show that confinement effects can easily be overcompensated by attractive protein–lipid interactions. The latter lead to a slight destabilization of the protein. No pressure-induced unfolding of the protein occurs up to 16 kbar.