Studies On Lipid Transmembrane Asymmetry Using Sum Frequency Generation Vibrational Spectroscopy

As an essential biointerface,the cell membrane plays key roles for many cellular processes.Therefore,the biomedical researches and applications based on the cell membrane have been gradually increasing,showing the great and unique value of the cell membrane interface.The most significant feature of the cell membrane is transmembrane asymmetry,related to its phospholipids,proteins and glycans.Previous studies mainly focused on the protein transmembrane asymmetry while overlooked the equally critical lipid transmembrane asymmetry.The reason,on one hand,is the obvious importance of membrane proteins.On the other hand,the lipid complexes of the cell membrane are highly sophisticated,leading to the few suitable characterization techniques.Sum frequency generation(SFG)vibrational spectroscopy is a powerful analytical tool,capable of probing molecular-level surface/interfacial structures and dynamics with its intrinsic surface/interface selectivity in situ in real time.Under the electric dipole approximation,SFG,as a nonlinear optical process,is forbidden for the materials with the inversion symmetry but is allowed on the surfaces or at the interfaces where the inversion symmetry is broken.Accordingly,SFG is an appropriate method to study the lipid transmembrane asymmetry and its movement,flip-flop,in cell membranes.In this dissertation,we applied SFG to the in-depth researches on the lipid transmembrane asymmetry and its related factors,including lipid properties,ionic environment and membrane proteins,on the basis of versatile biomimetic phospholipid bilayer systems.From the obtained information of molecular structures and dynamics at the interfaces,we revealed several effects on the lipid transmembrane asymmetry and provided their molecular mechanisms,which can be important guidance on maintaining and regulating the lipid transmembrane asymmetry in the cell membrane.Part I:Lipid unsaturation effects on lipid transmembrane asymmetry.The lipid unsaturation is asymmetric in the inner and outer leaflets of the cell membrane.Its maintenance mechanism is still unknown.We chose several kinds of phospholipids with different unsaturation to prepare the lipid bilayers,where asymmetric bilayer structures based on lipid unsaturation can be studied.The lipids with high saturation were found to be difficult for lipid transmembrane movement,flip-flop,and facilitate the formation and maintenance of the lipid transmembrane asymmetry in the lipid bilayers.The potential mechanisms were proposed to explain the maintenance of the asymmetric lipid unsaturation in the cell membrane.These findings can also contribute to the regulation of the cellular lipid components and the construction of versatile liposomes in biomedical applications.Part II:Metal ion size-dependent effects on lipid transmembrane asymmetry.The role of lipids in the regulation of cellular ion homeostasis is very important but not fully appreciated.We used the metal ions with similar properties but different ion sizes to investigate their influences on the lipid transmembrane asymmetry and lipid flip-flop.The match relationship between the ion size and the lipid distance was revealed,which led to the significant decrease of the flip-flop rate for the lipid bilayer.When the ion size and the lipid distance were mismatched,the flip-flop process was accelerated and the lipid transmembrane asymmetry was significantly destroyed.This study highlights the ion size effects on the lipid transmembrane flip-flop rates,providing the inspiring clue for understanding the lipid function related to lipid ion channels and cellular ion homeostasis.Part III:Effects of distinct protein binding states on lipid transmembrane asymmetry.The binding states of annexin V at the cell membrane interface are really complicated and the dynamic changes of the bond annexin V molecules are unclear.We conducted the comparative study on the distinct calcium(Ca²⁺)-dependent and-independent annexin V binding behaviors at the prepared biomimetic lipid membrane.It was discovered that the initial Ca²⁺-independent binding went through a transition with annexin V reorientation to a more stable state upon adding Ca²⁺.These binding behaviors affected the molecular arrangement of the membrane but preserved the lipid transmembrane asymmetry.The study provided a unique molecular-level perspective on the ubiquitous annexin binding behaviors and a valuable example of membrane protein influences on lipid transmembrane asymmetry.Part IV:Effects of protein conformational transition on lipid transmembrane asymmetry.The controllable protein conformational transition is of great significance to the amyloid-like protein aggregation reseaches at the cell membrane interface.We selected lysozymes as a model protein to successfully achieve the protein conformational transition at the lipid membrane interface,under the regulation of Tris(2-carboxyethyl)phosphine(TCEP)molecules.Compared the lysozyme molecules before and after the conformational transition,the change of the lysozyme secondary structures,from the?-helices to the?-sheets,was discovered.Meanwhile,the lysozyme aggregations and the interactions between lysozymes and the lipid bilayers also changed.During the process of the lysozyme conformational transition,the lipid transmembrane asymmetry was still held in the lipid membrane.In the research,the potential pathway and the molecular mechanism of the amyloid-like protein aggregation at the cell membrane interface were proposed,which can also be a useful exploration for maintaining and regulating the lipid transmembrane asymmetry in cell membranes.In summary,the maintenance and regulation of lipid transmembrane asymmetry is the core of the dissertation.Using advanced SFG spectroscopy,several vital factors for lipid transmembrane asymmetry were studied comprehensively,from the molecular structures to dynamics,exhibiting their varied effects and potential mechanisms and deepening our understanding on lipid transmembrane asymmetry at cell membrane interfaces.These findings supplied the theoretical references and the application guidance on maintaining and regulating lipid transmembrane asymmetry,promoting the development of the SFG characterization method for biointerfaces to some degree.In the future,the biomedical applications of such discoveries could attract extensive attentions and make a long-term contribution,along with the continuous verification and modification in complex living systems.