Lec 32 Flashcards
What is the method of HDX
Add protien to d2o solution
The amide hydrogens in disordered/exposed regions , like loops, exchange quicker than the secondary structures
Then stop reaction by reducing pH and digest with enzyme that works at low pH
Then do mass spec to see what regions exhange quick and what region exchanged slow
In a HDX sequence diagram, why are there gaps
What do you do to get more coverage of the sequence
Because you cant detect these peptides in the mass spec
Some peptides don’t have the cleavage site so don’t get cleaved
If the experiment wasn’t done in reducing conditions, the peptides with disulfide bonds wouldn’t show up (because not unfolded)
Use more than one protease
Explain the N to C HDX diagram
So you did the HDX experiment at diff time intervals
The red shows very fast exhange (meaning more flexible region) and blue show slow exchange (meaning rigid region)
How do you validate a sequence of a protien you got from MSMS
Compare it against the database of sequences of that organisms of interest and see which matches your sequence
Why do we use low pH in HDX
the rate of hydrogen exchange gets much slower at pH 2.5
This stops the reaction so that there’s time for the protease to cleave the protien and to the mass spec
Do this to make sure we increase the coverage of the protien
If you have protiens at pH 7 and pH 2.6 and have helix A B and D
In helix B, in the ph 7 shows red but the pH 2.6 shows little bit green then red what does this mean
In comparison to higher pH, the lower pH has slowed down the exchange
But since it goes back to red it’s still pretty fast
This means the protien is still keeping its fold and very stable since not really changing its folding at diff pH
If you have protiens at pH 7 and pH 2.6 and have helix A B and D
In helix A, in the ph 7 shows blue but the pH 2.6 shows blue then reddish as more time goes on what does this mean
In comparison to the high pH, the lower pH speeds up as the reaction goes on
This means it has unfolded and is more exposed
Usually low pH slows the exchange rate, but in this case the helix got unfolded after being in low pH for a long time and has more exposed residue for exchange
This means it’s less stable
If you have protiens at pH 7 and pH 2.6 and have helix A B and D
In helix D, in the ph 7 shows red but the pH 2.6 shows little bit green then red
Why would low ph show higher exchange
This must mean that even at low pH there are still regions that are solvent exposed (flexible) and have high exchange
What is the order of HDX exchange
Why
loops > alpha helix > beta sheet
Loops don’t do h bonding with each other
The back bone of beta sheets are h binding with each other (less accessible)
In alpha helix, backbones are pointing out (more accessible)
Why does the four helix bundle show more exchange at low ph
Since it’s backbones amides are exposed it has more exchange and is less well ordered at low pH
What are the key difference in folded and unfolded protiens
Folded:
Few hydrophobic side chains exposed
Energetically the most stable conformations (lowest point in the funnel)
Unfolded:
Many hydrophobic side chains exposed
Lots of conformations with equal stabilities
What type of entropy is good
What do we have to be careful of
Increasing entropy is good
Need to know what system you’re talking about (closed vs open) , local (the protein), global (the system)
What is conformational entropy in regard to
The local entropy , entropy of the protien
What is the conformational entropy of folded proteins
Unfolded
Why
Low
High
Folded structures are constrained and adopt a single well ordered structure that’s lower in energy then other structures
But unfolded protiens can have many different conformations similar in energy to each other and are more flexible
Knowing that folding is lower conformational entropy, what does thsi mean
The less entropy (more order) means that protein folding is an unfavourable change (since want increased entropy)
What is protien folding driven by
What does this also drive
The hydrophobic effect
The interactions between protiens
If you want to knock out the binding of a protien to another region of itself or another protiens what would you target
Why
Knock out the hydrophobic interactions
This decreases the conformational stability which allows more water molecules to enter that region of the protien
This increases the local conformational entropy and decreases the entropy of the system.
Overall decrease in entropy
What is the problem with hydrophobic groups in unfolded protiens being exposed
What is the result and what fixes it
This causes aggregation through hydrophobic interactions
Causes protiens misfolding diseases like prions
Chaperones like Hsp70 and GroEL fix it
What is the result of higher conformational entropy in unfolded protiens
What fixes this
Kinetic traps (tapped in a misfolded intermediate state)
Which are caused by cis- trans peptide isomerization (proline in cis conf), or disulfide bond isomerization (incorrect disulphide bonds)
Peptidyl prolyl isomerase: concert the trans proline to cis or vise versa
Disulphides isomerase: make sure the correct cysteines form the disulfide bond
What is cotraslational folding
Posttranslational
Which is easier and why
The protien is folding while being made on the ribosome in vivo
After translated, In vitro folding like the haber/anfinsen experiment
Co translational because everything needed for the protiens to fold is already nearby in vivo
In vitro, you don’t have the cellular machinery that normally folds the protien
What are protiens folding Catalysts, give examples
The help the protiens from getting stuck in kinetic traps during folding
Peptidyl prolyl isomerase
Proteins disulfide isomerase
What is Peptidyl prolyl isomerase (PPiases)
Proteins disulfide isomerase where is it
Peptidyl prolyl isomerase: catalyze cis trans isomerization of proline by lowering the energy barrier of trans to cis, this helps speed up the slow step of trans to cis during folding
Proteins disulfide isomerase:
They catalyze disulphide bond formation or breakage
In periplasmic space in bacteria
In endoplasmic reticulum in eukaryotes
If you want to produce an antibody in e coli where do you produce it
Fab need disulfide bonds to fold so :
In the periplasmic space for the disulfide isomerase to help it fold
Or
Secrete it to be extracellular in oxidizing environment
This oxidizes and forms the disulfide bonds
What is the proportion of cis and trans proline in a denatured protien and why
20% cis and 80 % trans
This is because the protien is denatured and can randomly flip and alter the proline orientation
What is the proportion of cis and trans proline in a folded protien and why
What about other residues
What is the exception
95% trans and 5% cis
Since folded it more constrained so prolines can’t flip to cis and trans conformations
Other residues are always trans
If in a loop/flexible region, back to 80:20 since it can flip
What is the purpose of cis prolines in a protien
It causes a kink which can play an important role in the function of the protien
Can turn the enzyme on or off like a switch
Know how to distinguish between cis and trans prolines structure
Okay
