Investigation Of Single Protein Dynamics In Plasma Membranes With Single-molecule Fluorescence Method

Plasma membranes separate the intracellular environment from the extracellular,which enclose the cell.Plasma membranes are also the site of many vital activities.Membrane proteins are the main executors of plasma membrane life processes.The conformation changes of membrane proteins are closely related with their biological effects.Therefore,it is very important to understand the dynamics of membrane proteins in the membrane,especially the relative position changes of membrane proteins in the vertical direction(longitudinal direction).The order and directionality of plasma membranes are based on the asymmetry of membrane.Maintenance of biomolecules transmembrane is essential for the maintenance of the asymmetry of the membrane and the cell.However,since the thickness of the living cell plasma membrane is only about 5 nm,the observation methods are required to nanometer resolution,which is hard to achieve with traditional observation methods.At present,it is urgent to develop new fluorescence observation method to observe the vertical movement of biomolecules along living cell plasma membranes.The premise is that the method could distinguish the vertical movement of membrane proteins from the tangential movement caused by membrane fluidity.Our group previously developed an in vitro fluorescence method based on planar support membrane and a liposome-based single molecule fluorescence method in vitro,to observe the position and dynamics of the membrane protein in the artificially prepared phospholipid bilayer membrane.We developed a method called QueenFRET(Quenchers in Extracellular Environment FRET).The method based on F(?)rster resonance energy transfer,which between the fluorophore labeled on biomolecules and quenchers in extracellular environment.The QueenFRET method may achieve a nanometer even subnanometer level in the vertical resolution,which realize the real-time measurement of biomolecules dynamics on and near living cell plasma membranes.In this work,the principle of QueenFRET method was introduced.We selected the fluorescence donor molecules and quencher suitable for the system.The distance dependence of the fluorescence modulation was quantitatively predicted based on the computational simulation.The paper firstly focused on the motion of biomolecules of known movement to verify the feasibility and precision of QueenFRET.We applied QueenFRET to study phospholipids,which are kinds of small biological molecule on plasma membrane.We measured the thickness of living cell plasma membranes accurately,which proved that the proposed method could observe the position of biomolecules with subnanometer precision.We observed the flip-flop and membrane fusion process of single phospholipid molecules on the membrane,which proved that the proposed method could observe the vertical movement process of biomolecules on the membrane dynamically in real time.Subsequent study extended this method to observation of membrane proteins,which have more complex chemical structure and function.We quantitatively observed the programmed necrosis related protein(MLKL)and its phosphorylation on the membrane movement.The experimental results demonstrating that this method is also suitable for the movement of protein sites on intracellular surface of plasma membranes.Above all,we demonstrate that QueenFRET can observe the vertical position of fluorescent molecules relative to the outer surface of the living cell plasma membrane,as well as the evolution and relaxation information of this distance over time and space.We applied QueenFRET to solve a biological problem: the directly transmembrane of antibacterial peptide LL-37,which caused by the selfassembly.A new mechanism of spontaneous transmembrane of peptides was proposed.The results also demonstrated that this method has unique advantages and wide applicability in the study of plasma membrane at the single-molecule level.The conclusion part summarized the advantages of the QueenFRET method and made prospects for follow-up research.QueenFRET open a new dimension in understanding the dynamics of living cell membrane proteins.By using QueenFRET,we can observe the diffusion on the biomolecular membrane while determining its depth and orientation relative to the surface of the biofilm in real time.These informations provide a better understanding of the motion scenes.of biomolecules within the membrane.It might be expected that this method will be widely used in the study of membrane biomolecular mechanism.