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
What happens if PAPD is gone
The complemtation beta strand isn’t present for papK
So papK has exposed hydrophobic residues and aggregation happens
In the fim system what is the homolog to papK and papD
PAPD= FimC
papK= FimA
In bacterial protiens, what is the general scheme of protection for folding
70 % of the time only need trigger factors
30% of the time DNAK and DNAJ try to fold the protien themselves , if that doesn’t work they deliver the protien to the GroEL groES chaperonin system
The protiens refold n the chamber
In eukaryotic protiens, what is the general scheme of protection for folding
Have HSP70 and HSP40 (DNA K AND J homologues) that try to fold the protien
If that doesn’t work the chaperonin hsp90 (uses atp) tries to fold it
Trigger factors don’t need ____ and are _____
Atp
Ribosome binding
What domains does the trigger factor have and what does this mean
Has a ppiase domain, meaning it also has PPiase activity in addition to being a chaperone
And a ribosome binding domain
What is Hsp70 called in E. coli
DNA K
What are the characteristics of the Hsp70 system
It’s atp dependent
Has Hsp70 and hsp40
In bacteria: Called dnak and dna J and also has GrpE
What is grpE
It main job it to take over a nucleotide : is a nucleotide exchange factor
Requires ATP and works with the groEL and GroES chaperonins
What is the structure of the HSP70 (DNAK)
Has a lid peptide binding domain
And an atpase domain (A region that binds adp / atp)
Both domains connected by a flexible linker
What is the open and closed state of the DNAK
With adp bound it’s closed and the lid domain blocks substare binding
With atp bound the lid open and the substrate binds
Explain the HSP70/DNAK cycle
- HSP70 is bound to atp (open) and in this state has high kon/koff
- DNAJ (HSP40) is the first one to bind the misfolded protein then recruits is to Hsp70
- Once the misfolded protien bind to hsp70, DNAJ and a phosphate are released
- Hsp70 now in closed adp state with low kon/koff and allows protien to fold (active folding)
- After folding (7-10 sec), GrpE releases the adp (but not open yet)
then atp binds and opens the Hsp70 complex so the folded protien can leave
Why is Hsp70 not considered a chaperonin but an active chaperone
not chaperonins because doesn’t fully enclose peptide
Uses atp
After the Hsp70 system what happens if protien still not folded
The protien get sent to the chaperonins
What is the restriction of dna J and k binding to a protiens that’s being made
They only bind to small 7 residue long hydrophobic patches
If bigger then that it’s a problem because cant have multiple dna J/k binding to the protein
Is there more DNAK/J than ribosome in the cells?
Yes, 100 fold more
Does DNAK help protiens smaller that 20-30kDA?
No , trigger factors can fix small protiens
What is special about the lid domain in the crysta strucure of the HSP70
Only when there is atp is the lid domain actually structured , waiting for something to come so it can immediately close
When nothing in the hsp70 (no atp or adp), the lid domain is flying all over the place until atp comes to make it structured
