Lec 33 Flashcards
In order for a protien to be folded properly what conf does proline have to be in
When folded, the prolines in the protien are only cis
Unfolded states can be cis and trans then they try to reach the cos folded state
Is the conversion of a peptide bonds (which has partial db characteristic) from cis to trans favourable increase comparison to a single bond
What does this mean
No it is highly unfavourable and has a high activation energy barrier
The rate of conversion from trans to cis is slow
What speeds up the isomerization of a protien from cis to trans
If the peptide is loosely structure and a t room temp
Lost some db character
What is the rate limiting step in folding of protien
The isomerization from trans to cis
The folding in a denatured trans to folded cis is slower than denatured cis to folded cis
How can trp flourense show the cis to cus and trans to cis fluor of the protien
Measuring At 320 nm shows the amount of total folded protien
The signal increases as the proteins fold
Initially it’s very fast because the already cis denatured protiens are folded to cis protein
But then increases slower because trans denatured to cis happens
Where are PPiases found
What organisms have them
On ribosomes, ribosome have the PPiase domain
They exist in all organisms
What do protien disulfide isomerase (PDI) help with
Since misparing of cysteine residues can cause protiens to be trapped In non native conformations
The PDI untrap them by breaking and reforming correct bonds
What is the major PDI in E. coli
DsbC
How does DsbC work
The protien is misfolded due to incorrect disulphide bond
DsbC react with and breaks the incorrect bond
Protien now unfolded can refold and form the correct bond
Why are DsbC located close to the membrane
When the protien is secreted out of the membrane it form these disulfide bonds
So that they can fix the potential misformed disulfide bonds of the protien as it’s being secreted out of the membrane it form
What another way to break the disulfide binds and let the protien refold properly
What is the downside
Add small amount reducing agent, this is slow (minutes to houre)
Using PDI with small amount reducing agent is fast (seconds)
Mutation in PDI causes
Disease because more misfolding
How many disulfide bonds in the Kringle domain and how many Kringle domains in plasminogen
How many in PAN
How many in protease
How many disulfide binds in total
What does this mean
3 per Kringle domain and 5 of them
2
6
23
So really has complex folding and needs correct disulfide bonds to fold properly
Where would you expect to see a protien with high amount of disulfide bonds
Secreted, extracellular, since need oxidizing environment
If one protien misfolded what happens
Causes others to misfold, hydrophobic residues exposed
More aggregation
Is aggregation common in folded protiens
Mo
What are Molecular chaperones
How are they expressed
They bind the misfolded state of the protiens TEMPORARILY (so have low affinity) to prevent aggregation, but leave to let the protien fold properly
Either constitutively expressed (there all the time for housekeeping)
Or stress induced : upregulated when there is heat or chemical stress causing more unfolded protiens than normal (like Hsp70)
What are the two types of chaperones
Passive (no atp needed): reduce the chance of misfolding but don’t actually control how they fold
Active (need atp)
What are the two ways that aggregation and misfolding are handeled by
chaperones in bacteria, yeast , mammals
and chaperonins
In Bacteria: trigger factor which is a ribosome accosiated chaperone which is already bound to the ribosome as the polypeptide is being made
Yeast: ssb it the trigger factor
Mammals: NAC complex is the trigger factor
Make sure that hydrophobic patch its t open for other protien to aggregate
Or chaperonins (if chaperone don’t work) fully enclose the protien in a compartment after it’s made and let it fold using atp
What is special about the folding of membrane protiens
They are largely hydrophobic and only fold once they’ve crossed the membrane
so one chaperone is attached to the unfolded poly peptide until it reaches the translocon
Then another chaperone waits on the other side of the membrane and the proton gets inserted into the membrane
What are the substrate specific chaperones
They target specific polypeptides
Ex. PAPD with is involved in pilus protien formation in bacteria
What are the general chaperones
Broad range of targets
Ex.
The trigger factor NAC
PFD (active chaperone)
Hsp70 family
Where does the PAPD protien in pilus in bacteria come from
The pilus formation is very complex and made up of many different protiens
The PAPD is a specific chaperone that is part of the pilus
How does PAPD work
How does it interacting with its substrate
Forms specific interactions with the pilin subunits
Bind to papK (a pilin subunit) to prevent aggregation before pilus assembly
Only when papK is around, PAPD has a complimentary beta sheets to allow interaction and binding
