PH induced reorganization of protein-protein interface in liposome encapsulated Ferritin at air/fluid and fluid/solid interfaces

Abstract

This study reports the pH-dependent assembly - disassembly of the iron storage cage protein ferritin (HSFr), in solution and enclosed in Giant vesicles (GUVs) of DOPC at air/buffer and fluid/solid interface. The surface pressure-area isotherms of films of lipids with the protein at air/buffer interface indicate that there is a disassembly ↔ assembly for pH shift from 7.0 to 2.0 back to 7.0, with a corresponding decrease ↔ increase in the average area/lipid. This disassembly and reassembly processes of ferritin-apoferritin show a pseudo reversible nature. Time resolved fluorescence of the protein in GUVs of mixed lipids of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (NBDPE) with DOPC revealed that the confined protein shows longer lifetimes than the free molecules. CD spectroscopy used to study the conformational preference of protein in the GUVs shows that localization of the protein does not introduce drastic changes in the structure of the vesicles which is confirmed by the fluorescence anisotropy values. The differences seen in the steady state fluorescence intensities and the lifetimes showing the differing encapsulation efficiency of the protein at the 2 pHs are in agreement with our air/buffer interface results. Viscoelastic behaviour of GUVs with the protein from Quartz Crystal Microbalance with dissipation, showed clearly the differences in the protein molecular assembly processes for pH 2.0 and 7.0. Flow cytometry (FC) analysis of the GUVs at pH = 2.0 showed that 18% of total GUVs have HSFr, while at pH 7.0, it is much less (around 7%). This difference could be due to the protein being in the monomeric unfolded state at pH 2.0 with good mobility than at pH 7.0 and therefore can be located easily inside the GUVs. This approach has applications in protein engineering strategies.