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Molecular Microbiology > Lecture 1 OMP biogenesis > Flashcards

Lecture 1 OMP biogenesis Flashcards

1
Q

Name 4 characteristics of the inner membrane of Gram-negative bacteria

A
  • Phospholipid bilayer
  • Symmetric
  • Integral membrane proteins (interactions on either side of the membrane)
  • Peripheral lipoproteins (attached to the membrane, but only on one side)
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2
Q

Name 5 characteristics of the OM of Gram-negative bacteria

A 
- Outer membrane (not a phospholipid bilayer)
• Phospholipid inner leaflet
• LPS in outer leaflet
• Asymmetric (bc different leaflets)
• Integral membrane proteins  OMPs
• Peripheral lipoproteins
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3
Q

Characteristics of IMP?

A
  • are alfa-helical
  • Transmembrane helices (TMs) act as membrane anchors
  • (1 - 10 TMs)
    • AAs (amino acids) in TMs are hydrophobic and contact acyl chains of phospholipids
    • (~20AA/TM)
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4
Q

Characteristics of OMP?

A

• folded into a barrel
• even number of beta-strands (8-24)
• strands 10 to >40 amino acid residues
− difficult to predict (from genes)

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5
Q

What is the build of OMP and how are the side chains positioned?

A
  • Built op of anti-parallel beta strands (are amphipathic, both hydrophobic and -philic)
  • amino acid side chains stick out from surface of beta sheet. If side chains are hydrophobic they are at the outside, hydrophilic inside.
  • Extra domains (periplasmic) are always found at the N-terminus
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6
Q

What do the beta-barrels of OMPs look like?

A

• Mono- or multimeric
− often trimers
• small loops at periplasmic side
• Long loops extending from the cell surface
− often functional ( e.g. as binding site, or used in gating )

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7
Q

Why are OMPs able to pass the cytoplasmic membrane to the periplasm?

A

Sec-machinery considers OMPs as soluble proteins -> OMPs have amphipathic beta-strands, which are not that hydrophobic, and can pass the cytoplasmic membrane to the periplasm.

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8
Q

What proteins do not pass the membrane and are placed in the cytoplasmic membrane?

A

Proteins containing alpha helices that are hydrophobic are placed laterally in the cytoplasmic membrane. Would not pass the membrane.

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9
Q

Name examples of OMP’s

A

• Trimeric Porins
Channels for uptake small molecules/nutrients
• Specific transporters
E.g. FhuA (iron ion receptor), recognize specific molecules
• Enzyme for modification of substrate (LPS). No pore. Modification of substrate
Often shorter beta-sheets. Such close contact of inward side chains that water cannot go through anymore, no hydrophobic channel inside
• Enzyme that anchors OM to peptidoglycan. No pore.
E.g. OmpA

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10
Q

Via what secretion system are OMPs transported through the inner membrane to the OM?

A

Sec

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11
Q

How do OMPs cross the inner membrane via Sec?

A

• N-terminal signal peptide (hydrophobic alpha helical stretch for targeting). Remaining part is not that hydrophobic.  OMPs enter periplasm unfolded. Signal peptide is cut off. Chaperones keep them in a transport competent state and prevent aggregation in periplasmic space.

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12
Q

What are the two functions of chaperones in the periplasm?

A

• Prevent aggregation
− Shield hydrophobic parts when protein starts to fold. Hydrophobic site is not compatible with the hydrophilic periplasm.
• Assist in folding
− Barrel is formed in periplasm prior to insertion in OM

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13
Q

What chaperones help with OMP insertion? What is their function?

A 
Skp, SurA
• protect against preliminary degradation
> Encage the OMP
DegP
• Has protease activity, 
degrades unfolded OMPs
> part of rpoE stress response
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14
Q

Skp: characteristics?

A
  • Homotrimer
  • Binds unfolded OMPs
  • Does not bind folded OMPs
  • Forceps-like arms
  • Prevents aggregation of OMPs
  • No role in folding
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15
Q

SurA characteristics?

A

Survival protein A (SurA)
• When mutated (knock-out): less OMPs produced, less OMPs assembled into OM
• Binds peptides rich in aromatic residues (which you find at rings of beta-barrel structures)
• Binds preferentially unfolded OMPs (better recognition of aromatic peptides)
• Helps formation of beta-barrel structure
- Folding chaperone
- Contains “PPI” activity

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16
Q

What is PPI activity?

A

“Peptidyl-prolyl cis/trans isomerase”

Changes conformation of prolines, which have a cis or trans confirmation and can be found in protein backbone. Prolines often positioned in turns and kinks. Changes protein conformation.

You find prolines mostly in the short turns in the periplasmic loops.

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17
Q

When are chaperones upregulated when the cell encounters stress?

A

With stress that interferes with the integration of OMPs in the OM and stress that is related to the intergrity of the OM

18
Q

What happens when surA and Skp are knocked out together?

A
  • SurA and Skp can be knocked out, but NOT together.
    = Synthetic lethality ( type of genetic interaction where the combination of two genetic events results in cell death or death of an organism.)
    Needed for efficiency and protection.
19
Q

What happens when there is stress in the cell regarding rpoE plasmid?

A

RPoE = Sigma e stress = whether proteins accumulate in the periplasm/not fold correctly. Also induced when soluble periplasmic proteins are accumulating

20
Q

What is the response of RpoE?

A

Overproduction of DegP protease.

21
Q

What does DegP do and how?

A

DegP: periplasmic protease

Forms a ring-like structure of 4 monomers -> stack of rings (12-24) on top of each other.
Cavity: degradation of not properly folded OMPs
Also chaperone -> can assist folding of beta-barrels
[not present in all bacteria]

22
Q

What does the Bam-complex do?

A

Assists in the final folding of the beta-sheet into a beta-barrel. Inserts barrel into the OM.

23
Q

What kind of gene did they look for when searching the candidates for OMP insertase?

A
  • Conserved among Gram-negatives, mitochondria and chloroplasts
  • Must be an essential gene
24
Q

They found OMP85. Why was this a good candidate?

A

Essential
Located in OM
In operon with skp (OMP chaperone) and lpxA (LPS biosynthesis). Both linked to OM biogenesis

25
Q

OMP85: essential gene

> Could not be knocked out, how to study?

A

Construct a conditional mutant.

  1. Introduce inducible copy of omp85 gene on a plasmid.
  2. Knock-out chromosomal copy. Mutant carrying this knock-out is still viable because it has the plasmid that contains the WT gene
  3. Cells get depleted of Omp85 protein in absence of the inducer. When introducing cells to inducer-less medium, there is a window in which you can study them.
26
Q

How to check for correct OMP-assembly?

A

Folding of OMPs can be monitored by SDS-PAGE

• Heat-modifiability:
Take two samples of a cell envelope, one which is denatured by heating, one at room temperature. Monitor whether presence of Omp85 led to folded or unfolded OMP at room temperature and denatured cell envelope.

27
Q

Characteristics of OMP regarding denaturation & stability?

A

 beta-barrel very stable
 resists denaturation by chemical SDS at room temp.
 folded form runs faster on SDS-PAGE

28
Q

Depleted Omp85 protein lead to unfolded OMPs. Conclusion?

A

Assembly OMPs into the outer membrane requires Omp85, renamed: BamA (Bam: Beta-barrel Assembly Machine)

29
Q

BamA was found in complex with..?

A

With 4 peripheral lipoproteins. BamB-E. BamD also essential, other components not.

30
Q

What was an indication that Bam complex could push OM proteins towards the beta barrel?

A

Crystal structure; 2 structures. They had differently distributed POTRA domains, as if there was some kind of movement. (POTRA domains are beneath the beta barrel)

31
Q

What happens when deleting POTRA domains?

A

POTRA domains deleted from BamA
• deletion of all is lethal
• Decreasing number decreased efficiency of insertion
• Effect more prominent on larger OMPs (22-stranded barrels)

32
Q

Function of POTRA domains?

A

The lipoproteins include lots of beta-strands. Beta-strands align and interact with the pre-mature beta-barrel proteins: a process that is called beta-augmentation. Helps align beta strands next to each other so it helps to form the sheet.

33
Q

What does BamA do with the membrane to make the uptake of barrels easier?

A

When looking at the aromatic rings, the membrane looks as is it is ‘squeezed’ at the backside. In vitro folding of OMPs goes better when membrane is thin.

Thinner membrane less stable, more movement lipids
 easier for uptake barrels

34
Q

What else indicates something is in the lumen of BamA and then pushed into the membrane?

A

BamA has a lateral gate.
First and last strand of beta strand positioned differently than in normal beta sheet proteins and they are shorter. Open vs closed state.

35
Q

How did they find out the lateral gate is important for the process?

A

Cross-linking the gate by two cysteines
• Form disulfide bond.
Could this disulfide bond protein replace the normal protein?

Effect:
• BamA inserted in OM
• BamA not able to complement wt-BamA (wt-BamA gene controlled by Arabinose-inducibe
promoter)

36
Q

What is the role of POTRA domains regarding the lateral gate?

A

 POTRA domains hinge the lateral gate to close and open.

37
Q

What are the current models for OMP insertion?

A
  1. BamA assisted model
  • provides a dynamic lipid bilayer
  • OMP folds spontaneously POTRA domains
  • bc of lipid movement: insertion takes place
  1. BamA-budding

Lateral gate is the place for beta-barrel folding and sideways translocating into the lipid bilayer

  1. Barrel-elongation

POTRA domains receive OMP, bring beta-strands into contact with the lateral gate, which helps to fold the sheet into the final barrel confirmation, then it is pushed into the OM.

38
Q

How is the process of OMP insertion energized?

A

interaction of the OMP + BamA = confirmational change. Lower folding energy -> difference in energy = used to help bamA change confirmation and push protein in lipid bilayer

39
Q

How does BamA recognize an OMP?

A

C-terminal targeting motif is important (deletion = aggregation in periplasm) in OMP (aromatic residues at the end, sort of conserved sequence)

40
Q

You can show pore activity with BamA conductivity assay. How does this work?

A

Two compartments: small hole in between. Lipid bilayer shutting off hole. Two electrodes: try to run a current, hydrophobic barrier prevents formation of a current. Then, to one compartment you can add the BamA protein, sometimes it will spontaneously insert itself into the lipid bilayer. Then you get a pore when unfoled OMP with signal sequence is added: current will run. Sometimes channels open and close: fluctuation in current. Pore does not open when adding OMP without signal sequence.

41
Q

Overview

A
  1. Integral OMPs are mostly beta-barrels
  2. Folding/assembly starts in periplasm
  3. Correct folding helped by chaperones
  4. BamA has role in membrane insertion and targeting
    • lateral gate
    • POTRAs
    • Lipid deformation
  5. C-terminus OMP contains Bam-targeting motif

Molecular Microbiology (12 decks)

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