Single-Molecule Force Spectroscopy Study Of ? Hemolysin

Pore-forming protein is a type of protein oligomer with transmembrane channel,which is formed by water-soluble monomer self-assembling on the cell membrane.The process of pore-forming is defined with four states including water-soluble monomer,membrane-bound monomer,prepore and pore.Pore-forming protein plays an important role in organism survival,metabolism and immune,which is secreted by bacteria or mammal.The transmembrane channel in bacterial outer membrane is beneficial to transfer nutrition from the environment.In contrast,many pore-forming protein produced by pathogenic bacteria are cytolytic specie to erythrocytes,leading to illness.When the cell is infected with inflammatory factors,the immune system is activated and releases pore-forming protein as an effector protein.Then,it assembles to form a membrane pore leading to cytolysis.Lytic cell exposes internal inflammatory factor,then clean up by immune cell to defend pathogen.Atomic force microscopy(AFM)is widely used to observe the topology change of protein in physiological conditions.With the development of fast scanning AFM,imaging time for one figure reduces to hundreds of milliseconds,which is essential to investigate pore-forming protein self-assembly.However,researchers rarely focus on monomer-membrane binding and the mechanism of monomer oligomerization.?-hemolysin(?HL)belonging to pore-forming protein is secreted as a water-soluble monomer by S.aureus,and binds to erythrocyte forming the transmembrane channel which leaks out low molecular weight molecules from cell to dead.?HL is a toxic protein for mammals according to previous reports.Therefore,atomic force microscopy-based single molecule force spectroscopy(AFM-SMSF)is used to investigate the self-assembly of ?HL monomer on the cell and its stability.In chapter 1,I first introduce the mechanism of self-assembly and classification of pore-forming protein,then focusing on ?HL,whose structure and function are described in detail.Meanwhile,the applications of AFM imaging and AFM-SMSF in membrane protein are displayed.In chapter 2,the image of ?HL obtained by AFM is shown under each state,in which the topology of ?HL monomer is different from its polyprotein.AFM imaging shows ?HL oligomer is successfully reconstituted into a bilayer,which is consists of seven protomers.SMFS is used to stretch the polyprotein constructed from a monomer,and force-distance curves showed several unfolding peaks.To detect the interaction between ?HL and membrane,?HL monomer immobilized on the AFM tip contacts the membrane at different loading time,retracting to record the unfolding events.In this experiment,it is amazing that water-soluble heptamer is stable.So SMFS is used to stretch water-soluble heptamer and membrane-bound heptamer to obtain the unfolding peaks.The number of amino acid for each peak are calculated and identified by steered molecular dynamics simulation.The kinetic parameters is obtained from different pulling speed,and showing membrane-bound heptamer is the most stable comparing to water-soluble heptamer and monomer.In chapter 3,we discovered the major factor for the highly stable membrane-bound?HL.In the crystal structure,stem and rim domain interact directly with the membrane.For the stem domain,the unfolding pathways of the mutants with pairwise removal of amino acid from stem domain are shown.To investigate the stability of stem domain mutants,the kinetic parameters as an evaluation criterion are compared and obtained at different pulling speeds.Rim domain is the other region close to the bilayer.The key residues of the two rim domain regions are identified by molecular dynamic simulation and mutated to glycine for confirmation.Comparing the unfolding pathways and the kinetic parameter from loading rate experiments shows the rim domain influences the stability of ?HL.As a result,stem and rim domain play a role in the ?HL stability and influence each other,and the two regions of the rim domain also have an interplay.In chapter 4,we investigated the interaction between ?HL and membrane.AFMSMFS is used to stretch ?HL in sodium deoxycholate(DOC),DPh PC bilayer with 2M Na Cl to decrease the electrostatic force and hydrogen bond,and in DPh PC bilayer containing 20%DPPS or 10% Lyso PC,showing distinct unfolding result.The results indicate that DOC is similar to DPh PC for ?HL stability.The interaction force between?HL and membrane contains electrostatic force,hydrogen bond and hydrophobic interaction.The last chapter is the summary and prospect of the study of ?HL.