Mechanisms Of Protein Unfolding And Its Impacts On The Structure And Phase Behavior Of Phospholipid Membrane

Protein-membrane interactions widely exist in organisms and they exert influenceson not only protein structures, but also the morphology, structure, and phase behaviorsof cell membranes. More importantly, proteins have highly-ordered folded structures,which can undergo complex structural changes such as unfolding and aggregation.Therefore, to study the influences of proteins on the structure and phase behavior of cellmembranes during the unfolding process is helpful to understand the mechanisms ofprotein-membrane interactions in organisms from a deep insight of physical chemistry.We constructed our research system with positively charged proteins (e.g.lysozyme) and negatively charged lipid membranes (DPPC-DOPG) which interactswith each other through electrostatic interaction. Calorimetry and thermal analysis(differential scanning calorimetry and isothermal titration calorimetry), spectroscopy(infrared spectroscopy, circular dichroism spectroscopy, and fluorescence spectroscopy),transmission electron microscopy, and synchrotron X-ray scattering have beenemployed to study the interactions between proteins and phospholipid modelmembranes. Based on our results on protein unfolding mechanisms, we further studiedthe protein structural changes after bound to lipid membranes and the influences on thelipid phase separation and membrane repeat distance exerted by membrane-boundproteins during their unfolding processes. These studies extend our understandings onprotein-membrane interactions from protein static structures to dynamic structuralchanges, and open a new broad window for us to probe into the protein-inducedstructural and phase behavior changes of lipid membranes.Throughout our studies, focus has been on the protein structural changes. A noveltemperature controlling method “interruption-incubation protocol” was proposed andapplied in calorimetric and infrared spectroscopic experiments to investigate the proteinunfolding cooperativity. The thermodynamically stable intermediates in the proteinunfolding process have been detected. For the electrostatic binding of lysozyme on lipidmembranes, we found that the most severe protein structural changes take place at anintermediate binding density by deep analyses of the protein unfolding thermal peaksand protein secondary structures. The underlying mechanism that structures of membrane-bound lysozyme are mediated by the two-dimensional binding density wasunraveled. Protein can induce lipid phase separation upon binding and this phenomenonwas intensively studied in our work. We found that during the unfolding processes,proteins rearrange their structures to achieve a better interaction with membranes andlipid lateral redistribution was induced by these specially unfolded membrane-boundproteins. Further efforts have been made to study the protein induced variation of lipidrepeat distance. It was found that arrangements of lipid head groups during phasetransition induce the refolding of membrane-bound lysozyme and finally lead to theswelling of lipid multilamellar membranes. The unfolded proteins, having induced thelipid phase separation, can “fasten” the lipid multilamellar membranes and keep therepeat distance constant. We attributed the structural changes of membranes toprotein-induced lipid phase behavior changes.