Hemolysin Pore

The expression of the a-hemolysin pore protein from S. aureus inside the vesicle solved the energy and material limitations the reactor could sustain expression for up to four days. 

Because DNA translocation events have main power spectral densities with a bandwidth of 10 kHz and R is also negligible compared to Rj, is approximated to Ri / 1 + ]2%f-Cj Rj ). As a result, at low frequencies, can be simplified to AkT/Rj, which is proportional to the conductance of the nanopore. Nanopore flicker noise is given by (a x F)/ (Nc xf). Here, I, a, and are the direct current, the Hooge parameter, and the number of charge carriers [18], respectively, all of which are associated with the KCl buffer concentration and nanopore material. To increase Rj, a nanopore with a narrow diameter should be adopted. For instance, the a-hemolysin pore with a limiting diameter of 1.5 nm has a resistance of 3 GO, in 0.3 M KCl or 1 GQ in 1 M KCl. 

Figure 1.5 Sketches of (a) the a-hemolysin pore and (b) solid-state nanopore used in single-molecule electrophysiology experiments.

Figure 11.2 Structure of the M pA pore and its mutants. The vestibule is more repulsive for a negatively charged polymer and the pore is shorter in comparison with a-hemolysin pore. (Adapted from Butler, T.Z. et al., Proc. Nall. Acad. Sci. USA, 105, 20647, 2008.)...

Wong, C.T.A. and Muthukumar, M., 2010. Polymo" translocation through a-hemolysin pore with tunable polymer-pore electrostatic rntCTaction, /. Chem. Phys., 133, 045101. 

Detection of ionic current fluctuation through an a-hemolysin pore created by a blockage of a pore by a strand of synthesized DNA... 

Staphylococcal a-hemolysin is another widely studied pore-forming toxin. It is used by infectious bacteria to perforate host animal cells by a mechanism that is distinct from that of gramicidin. Several aspects of the stmcture and function of this heptameric protein complex have been smdied. 

Aeromonas hydrophila is a bacterium that causes diarrheal diseases and deep wound infections. These complications arise due to pore formation in sensitive cells by the protein toxin aerolysin. Proteolytic processing of the 52-kD precursor proaerolysin (Figure 10.34) produces the toxic form of the protein, aerolysin. Like a-hemolysin, aerolysin monomers associate to form a heptameric transmembrane pore. Michael Parker and coworkers have proposed... 

Proteolysis of this precursor yields the active form, aerolysiu, which is responsible for the pathogenic effects of the bacterium in deep wound infectious and diarrheal diseases. Like hemolysin, aerolysiu monomers associate to form heptameric membrane pores. The three /3-strands that contribute to the formation of the heptameric pore are shown in red. The N-terminal domain (residues 1-80, yellow) is a small lobe that protrudes from the rest of the protein. 

Song, L., Hobangh, M., Shnstak, C., et al., 1996. Structure of staphylococcal o -hemolysin, a heptameric transmembrane pore. Science 274 1859 -1866. 

In reconstitution experiments, the self-assembly of the pore-forming protein a-hemolysin of Staphylococcus aureus (aHL) [181-183] was examined in plain and S-layer-supported lipid bilayers. Staphylococcal aHL formed lytic pores when added to the lipid-exposed side of the DPhPC bilayer with or without an attached S-layer from B coagulans E38/vl. The assembly of aHL pores was slower at S-layer-supported compared to unsupported folded membranes. No assembly could be detected upon adding aHL monomers to the S-layer face of the composite membrane. Therefore, the intrinsic molecular sieving properties of the S-layer lattice did not allow passage of aHL monomers through the S-layer pores to the lipid bilayer [142]. 

The prototype of a small pore-forming toxin is the S. aureus a-toxin, also called ct-hemolysin, that has been extensively investigated hy Bhakdi and coworkers. Monomers of ct-hemolysin (33 kDa) hind to the surface of erythrocytes, and after lateral diffusion within the lipid hilayer, seven monomers oligomerize to form pores in the cell membrane. The ct-hemolysin forms mushroom-shaped pores with an outer diameter of lOnm and an inner diameter of approximately 2.5 nm. Small molecules can pass through the pore and diffuse into/out of the cytosol, along with water. As a consequence of such movement, cell homeostasis is greatly disturbed and pushed into an unhealthy state. In animals, the a-hemolysin represents a major virulence factor of S. aureus which causes hemolysis as well as tissue destruction. ... 

Bacterial pore-forming proteins, such as oc-hemolysin, secreted by Staphylococcus aureus can be modified so that pore formation is activated by chemical, biochemical, or physical triggers. Such hemolysins, when targeted to tumors, could increase tumor permeability, and hence susceptibility to various cytotoxic drugs (78). 

Fig. 10 Model protocell systems, a An encapsulated polymerase (polynucleotide phos-phorylase) can synthesize RNA from nucleoside diphosphates such as ADP [79,80], b RNA can be synthesized by a template-dependent T7 RNA polymerase [83], c Proteins such as green fluorescent protein (GFP) can be synthesized by an encapsulated translation system [84], If mRNA coding for hemolysin is also present, the hemolysin forms a pore in the lipid bilayer. Amino acids then permeate the bilayer, and protein synthesis can continue for several days [85]...

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