Studying the structural organization of non-membranous protein hemoglobin in a lipid environment after reconstitution

In our current work we have developed a supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer with embedded hemoglobin, reconstituted via detergent-mediated method. Microscopic studies revealed that the hemoglobin molecules could be visualized without any labelling agents. The reconstituted proteins assemble themselves as supramolecular structures to adapt to lipid bilayer environment. The nonionic detergent, n-octyl-β-d-glucoside (NOG) used for insertion of hemoglobin played an important role in formation of these structures. When concentrations of lipid, protein and detergent were raised by four folds, we observed phase separation by protein molecules within bilayer via protein-protein assembly. This phase separation process exhibited extremely slow kinetics to form large stable domains with correlation times in the order of minutes. Confocal Z-scanning images showed that these supramolecular structures generated membrane deformities. UV-Vis, Fluorescence and Circular Dichroism (CD) measurement indicated minor structural change to expose the hydrophobic regions of the protein to adjust the hydrophobic stress of the lipid environment whilst Small Angle Neutron Scattering (SANS) results indicated that the hemoglobin molecules retained their overall tetrameric form in the system. In conclusion, we state that this investigation allowed us to closely inspect some rare but noteworthy phenomena like the formation of supramolecular structures, large domain formation and membrane deformation etc.