Lec 32

Question 1

What is the method of HDX

Answer 1

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

Question 2

In a HDX sequence diagram, why are there gaps

What do you do to get more coverage of the sequence

Answer 2

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

Question 3

Explain the N to C HDX diagram

Answer 3

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)

Question 4

How do you validate a sequence of a protien you got from MSMS

Answer 4

Compare it against the database of sequences of that organisms of interest and see which matches your sequence

Question 5

Why do we use low pH in HDX

Answer 5

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

Question 6

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

Answer 6

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

Question 7

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

Answer 7

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

Question 8

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

Answer 8

This must mean that even at low pH there are still regions that are solvent exposed (flexible) and have high exchange

Question 9

What is the order of HDX exchange

Why

Answer 9

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)

Question 10

Why does the four helix bundle show more exchange at low ph

Answer 10

Since it’s backbones amides are exposed it has more exchange and is less well ordered at low pH

Question 11

What are the key difference in folded and unfolded protiens

Answer 11

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

Question 12

What type of entropy is good

What do we have to be careful of

Answer 12

Increasing entropy is good

Need to know what system you’re talking about (closed vs open) , local (the protein), global (the system)

Question 13

What is conformational entropy in regard to

Answer 13

The local entropy , entropy of the protien

Question 14

What is the conformational entropy of folded proteins

Unfolded

Why

Answer 14

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

Question 15

Knowing that folding is lower conformational entropy, what does thsi mean

Answer 15

The less entropy (more order) means that protein folding is an unfavourable change (since want increased entropy)

Question 16

What is protien folding driven by

What does this also drive

Answer 16

The hydrophobic effect

The interactions between protiens

Question 17

If you want to knock out the binding of a protien to another region of itself or another protiens what would you target

Why

Answer 17

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

Question 18

What is the problem with hydrophobic groups in unfolded protiens being exposed

What is the result and what fixes it

Answer 18

This causes aggregation through hydrophobic interactions

Causes protiens misfolding diseases like prions

Chaperones like Hsp70 and GroEL fix it

Question 19

What is the result of higher conformational entropy in unfolded protiens

What fixes this

Answer 19

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

Question 20

What is cotraslational folding

Posttranslational

Which is easier and why

Answer 20

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

Question 21

What are protiens folding Catalysts, give examples

Answer 21

The help the protiens from getting stuck in kinetic traps during folding

Peptidyl prolyl isomerase

Proteins disulfide isomerase

Question 22

What is Peptidyl prolyl isomerase (PPiases)

Proteins disulfide isomerase where is it

Answer 22

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

Question 23

If you want to produce an antibody in e coli where do you produce it

Answer 23

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

Question 24

What is the proportion of cis and trans proline in a denatured protien and why

Answer 24

20% cis and 80 % trans

This is because the protien is denatured and can randomly flip and alter the proline orientation

Question 25

What is the proportion of cis and trans proline in a folded protien and why

What about other residues
What is the exception

Answer 25

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

Question 26

What is the purpose of cis prolines in a protien

Answer 26

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

Question 27

Know how to distinguish between cis and trans prolines structure

Answer 27

Okay