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Bcem 431 > Lec 33 > Flashcards

Lec 33 Flashcards

1
Q

In order for a protien to be folded properly what conf does proline have to be in

A

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

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2
Q

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

A

No it is highly unfavourable and has a high activation energy barrier

The rate of conversion from trans to cis is slow

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3
Q

What speeds up the isomerization of a protien from cis to trans

A

If the peptide is loosely structure and a t room temp

Lost some db character

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4
Q

What is the rate limiting step in folding of protien

A

The isomerization from trans to cis

The folding in a denatured trans to folded cis is slower than denatured cis to folded cis

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5
Q

How can trp flourense show the cis to cus and trans to cis fluor of the protien

A

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

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6
Q

Where are PPiases found

What organisms have them

A

On ribosomes, ribosome have the PPiase domain

They exist in all organisms

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7
Q

What do protien disulfide isomerase (PDI) help with

A

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

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8
Q

What is the major PDI in E. coli

A

DsbC

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9
Q

How does DsbC work

A

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

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10
Q

Why are DsbC located close to the membrane

A

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

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11
Q

What another way to break the disulfide binds and let the protien refold properly

What is the downside

A

Add small amount reducing agent, this is slow (minutes to houre)

Using PDI with small amount reducing agent is fast (seconds)

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12
Q

Mutation in PDI causes

A

Disease because more misfolding

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13
Q

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

A

3 per Kringle domain and 5 of them

2

6

23

So really has complex folding and needs correct disulfide bonds to fold properly

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14
Q

Where would you expect to see a protien with high amount of disulfide bonds

A

Secreted, extracellular, since need oxidizing environment

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15
Q

If one protien misfolded what happens

A

Causes others to misfold, hydrophobic residues exposed

More aggregation

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16
Q

Is aggregation common in folded protiens

A

Mo

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17
Q

What are Molecular chaperones

How are they expressed

A

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)

18
Q

What are the two types of chaperones

A

Passive (no atp needed): reduce the chance of misfolding but don’t actually control how they fold

Active (need atp)

19
Q

What are the two ways that aggregation and misfolding are handeled by

chaperones in bacteria, yeast , mammals

and chaperonins

A

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

20
Q

What is special about the folding of membrane protiens

A

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

21
Q

What are the substrate specific chaperones

A

They target specific polypeptides

Ex. PAPD with is involved in pilus protien formation in bacteria

22
Q

What are the general chaperones

A

Broad range of targets

Ex.
The trigger factor NAC
PFD (active chaperone)
Hsp70 family

23
Q

Where does the PAPD protien in pilus in bacteria come from

A

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

24
Q

How does PAPD work

How does it interacting with its substrate

A

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

25
Q

What happens if PAPD is gone

A

The complemtation beta strand isn’t present for papK

So papK has exposed hydrophobic residues and aggregation happens

26
Q

In the fim system what is the homolog to papK and papD

A

PAPD= FimC

papK= FimA

27
Q

In bacterial protiens, what is the general scheme of protection for folding

A

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

28
Q

In eukaryotic protiens, what is the general scheme of protection for folding

A

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

29
Q

Trigger factors don’t need ____ and are _____

A

Atp

Ribosome binding

30
Q

What domains does the trigger factor have and what does this mean

A

Has a ppiase domain, meaning it also has PPiase activity in addition to being a chaperone

And a ribosome binding domain

31
Q

What is Hsp70 called in E. coli

A

DNA K

32
Q

What are the characteristics of the Hsp70 system

A

It’s atp dependent

Has Hsp70 and hsp40

In bacteria: Called dnak and dna J and also has GrpE

33
Q

What is grpE

A

It main job it to take over a nucleotide : is a nucleotide exchange factor

Requires ATP and works with the groEL and GroES chaperonins

34
Q

What is the structure of the HSP70 (DNAK)

A

Has a lid peptide binding domain

And an atpase domain (A region that binds adp / atp)

Both domains connected by a flexible linker

35
Q

What is the open and closed state of the DNAK

A

With adp bound it’s closed and the lid domain blocks substare binding

With atp bound the lid open and the substrate binds

36
Q

Explain the HSP70/DNAK cycle

A
  1. HSP70 is bound to atp (open) and in this state has high kon/koff
  2. DNAJ (HSP40) is the first one to bind the misfolded protein then recruits is to Hsp70
  3. Once the misfolded protien bind to hsp70, DNAJ and a phosphate are released
  4. Hsp70 now in closed adp state with low kon/koff and allows protien to fold (active folding)
  5. 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

37
Q

Why is Hsp70 not considered a chaperonin but an active chaperone

A

not chaperonins because doesn’t fully enclose peptide

Uses atp

38
Q

After the Hsp70 system what happens if protien still not folded

A

The protien get sent to the chaperonins

39
Q

What is the restriction of dna J and k binding to a protiens that’s being made

A

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

40
Q

Is there more DNAK/J than ribosome in the cells?

A

Yes, 100 fold more

41
Q

Does DNAK help protiens smaller that 20-30kDA?

A

No , trigger factors can fix small protiens

42
Q

What is special about the lid domain in the crysta strucure of the HSP70

A

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

Bcem 431 (33 decks)

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