Probing Single-protein Dynamics On Membranes With Liposome-based Single-molecule Fluorescence Attenuation Method

Biological membranes are selectively permeable barriers for cells in maintaining homeostasis.They also act as sites for multiple biochemical reactions.The functions of membrane system rely on various membrane proteins.Probing the position of proteins on membranes,especially the position changes relative to the membrane surface,is crucial for understanding the function and mechanism.Liposomes,which maintain the fluidity of the phospholipids and has adjustable curvature,are ideal bio mimic systems to study the functions of membrane proteins.However,the thickness of the membranes is typically only 3-5 nm.In order to probe the positions of membrane proteins relative to the membrane surface of a liposome,the resolution of the measurements is required to be nanometers or even sub-nanometers,which was difficult for traditional researching methods.In this work,we developed a liposome-based single-molecule fluorescence method,which was named Lipo FRET,to probe the positions and dynamics of a single protein on the membrane of a liposome in real time.The method takes the principle of the fluorescence resonance energy transfer?FRET?between the fluorophore labeled on the membrane protein and multiple quenchers encapsulated in the liposome.Lipo FRET provides nanometer or sub-nanometer resolution in membrane protein dynamics studies.We had chosen suitable FRET donors and quenchers for Lipo FRET assay,and explored the quenching properties of the liposome system with numerical simulations.We also demonstrated the feasibility of Lipo FRET with fluorophore-labeled phospholipid,single-and double-strand DNA,and antimicrobial peptide LL-37.As the application of the method,we applied Lipo FRET to study the membrane interaction of?-synuclein,a Parkinson's disease related protein.Our study discovered new knowledge about the interaction between?-synuclein and lipid membranes.The results showed the central helix of?-synuclein stayed at the surface of the liposome membranes,while the acidic C-terminal tail was in the aqueous solution.The N-terminal transitioned slowly among several depths in the membrane,which was never been reported before.The impact of Ca²⁺on C-terminal conformation was also investigated.In addition,we made preliminary insight with Lipo FRET into the interaction between?-synuclein and VAMP2,which was associated with membrane fusion.In further study towards?-synuclein,we found the membrane interaction pattern of the protein changes as the protein concentration changes,which was associated with accelerated microscopic dissociation.The effect of Y39 phosphorylation of?-synuclein on the protein-membrane interaction was also examined by surface-induced-fluorescence-attenuation method,which would be studied with Lipo FRET in the future.These results demonstrated Lipo FRET as a powerful tool in studying membrane protein dynamics.Membrane-protein interactions remains a main subject in the field of biological membrane.It attracts much interest because of the importance of membrane system in cells and organisms.However,knowledge about the dynamics of membrane proteins in lipid bilayer is still poor because of the lack of powerful tools.The results about?-synuclein demonstrated that Lipo FRET was capable to detect position changes of a membrane protein in and out of the membrane.Lipo FRET is easy to implement.It does not require specific instruments.It is also straightforward to extend Lipo FRET to other applications,such as living cells,which is the future direction of our methods.We anticipate Lipo FRET will have more widespread applications in the field of biological membranes.