Membrane Insertion of Protein Toxins CD Flashcards
Name some types of toxins
Colicin A, Diptheria, Exotoxin A, Cholera, Tetanus
What are the domains in Colicin A?
outer memb translocation:Receptor Binding:Memb insertion
What are the domains in Diptheria Toxin?
Enzymatic:Memb insertion:Catalytic domain
Enz and Memb insertion are nicked and held together by a disulphide bond until they enter the cell
What is memb insertion/translocation promoted by?
Low pH and a trans negative memb potential
Inside cell normally -70mV
Memb is polarised
Colicin Memb insertion domain
Helical and soluble
All memb inserting domains are water soluble proteins
What is diptherias mode of action?
Recognises receptors in upper respiratory tract
Binds to host membrane
Membrane bound toxin enters by endocytosis (receptor mediated endocytosis)
Catalytic subunit is cleaved by held to the other subunits by disulphide bonds
Endosome vesicle acidifies and the disulphide bonds are reduced-reprotonates SS bond and breaks into thiol group
The TM domain facilitates the passage of the catalytic peptide through the vesicle membrane and insects into the cytoplasm of the target cell
The catalytic domain ADP Ribosylates elongation factor 2 (using a molecule of NAD)
Few molecules of EF2 floating around the cell so don’t nee many of theses to be knocked out by ADP-ribosykation
This halts protein synthesis and kills the cell
What is colicins mode of action?
Attacks other bacteria
Receptor binding domain targets a particular protein on target cell of a competing bacteria
Outer membrane translocation domain inserts
Membrane insertion domain is pulled across the the outer membrane and inserts into the interior membrane of the bacterium
Membrane potential is around -50mV of other bacteria which helps the membrane insertion domain to insert into the membrane which creates a pore so that ions and water leak out
Loss of membrane potential so target cell dies
What is unusual about the colicin membrane insertion domain?
Helical structure but it has typical properties of a water soluble protein
Hydrophobic aa mostly clustered in interior of protein
Polar/charged aa on outside
How can water-soluble proteins or domains spontaneously insert into the membranes and promise membrane translocation of catalytic domains?
Do the hydrophobic/hydrophillic properties of aa that dominate folding of soluble proteins apply to membrane proteins? YES
Water soluble protein can insert into membranes is not because of anything odd about the contribution of was to membrane protein insertion since the hydrophobicity scale is similar to what we already know
One clue to answering how a water soluble protein can insert into membranes comes from looking at some smaller peptide toxins like the bee venom melittin (small peptide)
Has some similarities to the bacterial toxin such that in solution this polypeptide forms a tetrameric bundle
What is the hydrophobicity order of aas?
FMILVYWCATGSPHQNEKDR
What can YidC do on its own?
Catalyse insertion of a small class of proteins with 1 or 2 membrane spanning helices like a hairpin Catalyses insertion of M13 Coat protein
How does the m13 coat protein get inserted into the membrane?
Is inserted by a leader/signal peptide with 2 helix domains which become cleaved by a leader peptidase only when it has been correctly inserted into the membrane.
Single TM helical protein which agglomerates together in a regular array to form the coat of the m13 virus
How does the colicin membrane insertion umbrella model work?
It has amphipathic helices and 2 hydrophobic helices in the centre
Ampipathic helices find membrane surface, non polar parts face lipids
Membrane inserting domain sort of unfolds and inserts into the membrane
Insertion of 2 central hydrophobic helices with amphipathic relics on the membrane surface
Low pH causes protonation of Asp and Glu which breaks salt bridges and promotes helix unpacking, increases overall positive charge and forms a stronger interaction with the membrane potential
Breaks some of the cohesive forces that hold the water soluble domain together.
CD Expt exploring Colicin umbrella model
CD can see whether a protein has a properly folded core-can tell you about the inherent asymmetry of the structure
Add acid which is indicative of the umbrella model
alpha helical structure retained/unchanged haven’t lost secondary structure in helices - confirms model
Tryp residues in asymmetrical hydrophobic core give strong signal for a tightly folded protein as locked into this asymmetric core-induces asymmetry within the aromatic side chains, this is reduced in umbrella model when colicin inserts its membrane domains so it is not as nice and compact (more globular and unfolded so not in an induced asymmetric environment)/and acidification-consistent with model
How do you use membrane bound colicin A in experiment with bromine to look at umbrella model?
Fluorescence can be v strongly quenched by electron rich atoms such as bromine or iodine. The fluorescence spectrum of tryptophan in a solution of sodium bromide gives a very strong fluorescence because the tryptophans are sequestered and facing away from the solution so they don’t interact with bromine which would otherwise quench the fluorescence
You can make a membrane that has bromine incorporated by brominating across double bonds-a phospholipid with bromine in it and then make a bilayer membrane with all the brominated lipids. The fluorescence signal of colicin when its bound to these membranes you find that the tryptophan fluorescence is strongly quenched. These tryptophan are coming into contact with bromine in the membrane-also consistent with umbrella model where the protein unfolds and inserts two hydrophobic helices into the membrane
