NalP: Difference between revisions

Line 26:

In order to test the function of the alpha helix within the domain a test was done in order to compare results of uptake within the domain in the presence of the alpha helix and without the alpha helix. An easy way to test this was an antibiotic assay. By isolating colonies strictly of gram-negative bacteria with the alpha helix, NalPβ, and isolating colonies strictly of gram negative bacteria without the alpha helix, NalPβΔhelix, they could plate these separately. From there it was possible to compare susceptibility to antibiotics by placing the small circular tabs of antibiotics on the plate and measuring the difference in how effect the antibiotics was in penetrating the cell. The more penetration would show less growth inhibition or more sensitivity to the antibiotics. When the alpha helix was removed there was much more sensitivity to antibiotics showing that the removal of it leads to a more open pore.

== Integration of the Translocator Domain into Outer Membrane ==

Omp85 has been found in many studies to help integrate beta barrels into the outer membrane in order to allow the autotransporter to complete its duty. Do to the hydrophilic nature of the beta barrel’s hairpin loops on the extracellular side in NaIP it is impossible for it to breach the cell membrane that is highly hydrophobic. Research of how this occurs in Neisseria meningitides is ongoing and has not been discovered yet. Yet there are many implications that a protein named Omp85 is most likely the helper protein that facilitates this. The large hydrophilic loops on the auto transporter domain might act as a recognition signal for the Omp85 complex to encompass the end of the beta barrel. From here the Omp85 complex transports the translocator toward the cell membrane and is able to created a pore and place the beta barrel within the membrane, whilst avoiding that hydrophilic loops from directly coming in contact with the hydrophobic cell membrane.

Line 42:

Line 47:

The Hairpin Model is a much more likely model that could accommodate for the movement across the membrane. The hairpin model allows for a hairpin loop to be created in the passenger protein and as it passes through the end of the pore a hairpin loop there interacts with the passenger protein in order to create the folding. As the protein is folded it provides energy to pull the rest of the protein through. One major problem with this model is the fact that 2 strands must fit through the protein at once and with the alpha helix that is an impossible fit. The model describes a fix to this problem with the destruction of the alpha helix and recreation. When the alpha helix is nonexistent the hydrogen’s that typically face inward and interact with the alpha helix face toward one another or even outward which expands the barrel as well as making it much more flexible. This model suggests that folding and translocation are interdependent and happen simultaneously. The problem with this method is there is no mechanism for how the alpha helix is created or dismantled and where it goes.

=== Alternative Theory ===

One alternative theory argues that the beta barrel is actually not used as a protein secretion pore at all. As Omp85 compasses then end of the beta barrel it travels toward the cell membrane as if to place the autotransporter into the cell membrane yet instead of placing it, Omp85 continues through into the extracellular material. As the translocator is being carried toward the cell membrane it is able to pick up a passenger protein using its loose c-terminus end that would have faced inward toward the paraplasm. Then all three the, Omp85, translocator, and the passenger protein are all transported to extracellular material through the pore that Omp85 is able to create. Then they all dissociate away from one another, which frees the passenger protein. This is another possibility for how the translocator is able to transport passenger protein out of the cell yet changes the view of the translocator all-together. If this is in fact the way that passenger proteins leave the cell then NaIP is no a autotransporter at all. Autotransporter just as it sounds autotransports, meaning that the protein pulls itself through as it is folded on the opposite side of the cell. As plausible as this seams it would mean a major change in the way that this translocator protein is classified.

== Similar Structure in Other Proteins ==

Recent research has showed that there are possible conserved features to this pore and pores in other types of gram-negative bacteria. Autotransporters that also have the conserved structure of an alpha helix directly preceding the beta core include: AidaI of E. coli, BrkA of B. pertussis, Hap of Hemophilus influenzae and IgA protease and App of N. meningitidis. Much of the these proteins show low conservation within their alpha helixes yet they all have a long traversing alpha helix that leads into the 12 sheeted beta barrel. Due to the amount of research being done on the Neisseria meningitidis' NalPβ protein, its crystal structure is being used in order to model autotransporter secretion.

Navigation menu

Page actions

Page actions

Personal tools

Navigation

Search

Tools

@@ Line 26: / Line 26: @@
 In order to test the function of the alpha helix within the domain a test was done in order to compare results of uptake within the domain in the presence of the alpha helix and without the alpha helix. An easy way to test this was an antibiotic assay. By isolating colonies strictly of gram-negative bacteria with the alpha helix, NalPβ, and isolating colonies strictly of gram negative bacteria without the alpha helix, NalPβΔhelix, they could plate these separately. From there it was possible to compare susceptibility to antibiotics by placing the small circular tabs of antibiotics on the plate and measuring the difference in how effect the antibiotics was in penetrating the cell. The more penetration would show less growth inhibition or more sensitivity to the antibiotics. When the alpha helix was removed there was much more sensitivity to antibiotics showing that the removal of it leads to a more open pore.
+== Integration of the Translocator Domain into Outer Membrane ==
+Omp85 has been found in many studies to help integrate beta barrels into the outer membrane in order to allow the autotransporter to complete its duty. Do to the hydrophilic nature of the beta barrel’s hairpin loops on the extracellular side in NaIP it is impossible for it to breach the cell membrane that is highly hydrophobic. Research of how this occurs in Neisseria meningitides is ongoing and has not been discovered yet. Yet there are many implications that a protein named Omp85 is most likely the helper protein that facilitates this. The large hydrophilic loops on the auto transporter domain might act as a recognition signal for the Omp85 complex to encompass the end of the beta barrel. From here the Omp85 complex transports the translocator toward the cell membrane and is able to created a pore and place the beta barrel within the membrane, whilst avoiding that hydrophilic loops from directly coming in contact with the hydrophobic cell membrane.
@@ Line 42: / Line 47: @@
 The Hairpin Model is a much more likely model that could accommodate for the movement across the membrane. The hairpin model allows for a hairpin loop to be created in the passenger protein and as it passes through the end of the pore a hairpin loop there interacts with the passenger protein in order to create the folding. As the protein is folded it provides energy to pull the rest of the protein through. One major problem with this model is the fact that 2 strands must fit through the protein at once and with the alpha helix that is an impossible fit. The model describes a fix to this problem with the destruction of the alpha helix and recreation. When the alpha helix is nonexistent the hydrogen’s that typically face inward and interact with the alpha helix face toward one another or even outward which expands the barrel as well as making it much more flexible.  This model suggests that folding and translocation are interdependent and happen simultaneously.  The problem with this method is there is no mechanism for how the alpha helix is created or dismantled and where it goes.
+=== Alternative Theory ===
+One alternative theory argues that the beta barrel is actually not used as a protein secretion pore at all. As Omp85 compasses then end of the beta barrel it travels toward the cell membrane as if to place the autotransporter into the cell membrane yet instead of placing it, Omp85 continues through into the extracellular material. As the translocator is being carried toward the cell membrane it is able to pick up a passenger protein using its loose c-terminus end that would have faced inward toward the paraplasm. Then all three the, Omp85, translocator, and the passenger protein are all transported to extracellular material through the pore that Omp85 is able to create. Then they all dissociate away from one another, which frees the passenger protein. This is another possibility for how the translocator is able to transport passenger protein out of the cell yet changes the view of the translocator all-together. If this is in fact the way that passenger proteins leave the cell then NaIP is no a autotransporter at all. Autotransporter just as it sounds autotransports, meaning that the protein pulls itself through as it is folded on the opposite side of the cell. As plausible as this seams it would mean a major change in the way that this translocator protein is classified.
 == Similar Structure in Other Proteins ==
 Recent research has showed that there are possible conserved features to this pore and pores in other types of gram-negative bacteria. Autotransporters that also have the conserved structure of an alpha helix directly preceding the beta core include: AidaI of E. coli, BrkA of B. pertussis, Hap of Hemophilus influenzae and IgA protease and App of N. meningitidis. Much of the these proteins show low conservation within their alpha helixes yet they all have a long traversing alpha helix that leads into the 12 sheeted beta barrel. Due to the amount of research being done on the Neisseria meningitidis' NalPβ protein, its crystal structure is being used in order to model autotransporter secretion.