L6 - Late stages in protein folding

Question 1

What is the molten globule?

Answer 1

The experimental evidence (CD spectroscopy, NMR-based hydrogen exchange, rapid mixing) has shown that one significant kinetic intermediate is the molten globule

Its structure resembles that of an equilibrium molten globule formed by low pH (urea etc.)

Question 2

What separates the early and late stages of protein folding?

Answer 2

An intermediate

It is a molten globule – isn’t rigidly defined by the side chains & is still flexible – still fairly fluid it its interactions with its side chains

Only exists for a period of time before making the native protein

It already has a hydrophobic core

Question 3

How do we study the molten globule?

Answer 3

We can study the molten globule by having an intermediate urea concentration or changing the pH so the protein stays in the intermediate state

The MG is a broad example of what we expect – looks quite like the final structure, has a lot of the secondary structure & is a similar size but it doesn’t have the important final interactions which are characteristic of a well folded protein structure

Question 4

What do experiments from creating a stable ‘molten globule state’ under mildly denaturing conditions show?

Answer 4

Presence of substantial secondary structure

Absence of most of the specific tertiary structure associated with tight packing side chains

Dynamic feature of structure with motions on a time-scale longer than nanoseconds

Compactness of the molecule with a radius only 10-30% larger than the native state

Presence of loosely packed hydrophobic core that increases the solvent accessible hydrophobic surface that binds to ANS

Question 5

Detecting the molten globule intermediates using fluorescence

Answer 5

Extrinsic fluorescence (ANS) detects the unfolded intermediate (eg. the molten globule)

It inserts into the loose hydrophobic core & its fluorescence thus increases

It does not bind to unfolded or folded states

Question 6

Why does ANS not bind to unfolded or folded states?

Answer 6

Completely unfolded there’s no hydrophobic environment for the probe to dissolve into

When the proteins fully folded ANS cannot get in there

Only in the intermediate state can it bind

Question 7

Detecting the molten globule intermediates using circular dichroism

Answer 7

Can pick out other signals if you extend the wavelengths – they come from the side chains such as tryptophan & tyrosine

When the amino acid side chains get fixed in the 3D structure of the protein, we can see a signal

If the protein is MG or unfolded, then side chains don’t form a fixed conformation so we cannot see a signal from them

Question 8

What does the far UV & the near UV show in CD?

Answer 8

Far UV – secondary structure

Near UV – side chains

Question 9

Signature state of a native (folded) protein

Answer 9

• Far UV
• Near UV
• Strong Trp fluorescence
• No ANS fluorescence
• Cooperative unfolding

Question 10

Signature state of a MG protein

Answer 10

• Far UV
• No near UV
• Strong Trp fluorescence
• Strong ANS fluorescence
• No cooperative unfolding

Question 11

Signature state of an unfolded protein

Answer 11

• No far UV
• No near UV
• No Trp fluorescence
• No ANS fluorescence
• No cooperative unfolding

Question 12

Reason for slower folding of intermediates

Answer 12

Mostly complicated energy barriers
Trying to piece together very precise interactions

Here we will look at 2 examples which we do understand

• Proline isomerisation
• Disulphide bridge formation

But remember: most barriers to folding of intermediates are more complicated & do not involve the above

Question 13

Proline isomerisation

Answer 13

Peptide bonds in proteins are mainly trans
Thus amino acids are mostly in the right configuration already

Amino acid X-Proline peptide bond exists in cis form approx. 20-25% in unfolded peptide
In 25% of cases the proline will be in the wrong conformation so cannot go any further

Proline wants to be trans – will happen slowly by thermal displacement (random isomerization) – this would really slow down protein folding

Nature has developed enzymes called PPI which accelerate this process

Question 14

Peptidyl-prolyl-isomerase (PPI)

Answer 14

In the cell the ‘normal’ rate of folding seen in vitro can be accelerated by an enzyme which increases the rate of trans-cis isomerisation, PPI

Also called cyclophillins, members of this family bind the immunosuppressive drug cyclosporin A (CsA)

Question 15

Disulphide bridges

Answer 15

Disulphide bridges (as in Anfinsen’s experiment) are needed for some extracellular proteins to function properly

When more than 2 cysteins are present there is a chance that incorrect disulphide bridges occur during maturation of the protein on the secretory pathway

Need an enzyme which will rearrange the incorrect disulphide bridges

• ER & Golgi in eukaryotes – PDI
• Periplasm in E. coli – DsbA (gram -ive)
• Cell wall in Bacillus – Bdb (gram +ive)

Question 16

Disulphide bridges in E. coli

Answer 16

In E. coli secreted proteins are oxidised by DsbA & incorrect bonds rearranged by DsbC

• DsbA is recycled by DsbB, which is in turn reduced by quinols from respiration
• DsbC is recycled by DsbD & thiredoxin

2 steps here

1) Accelerating the formation of the disulphide bonds
2) If it gets it wrong then DsbC will increase exchange of bonds until they’re correct

Question 17

Disulphide bridges in yeast

Answer 17

A eukaryotic example

In yeast PDI does both jobs and gets recycled by proteins associated with the oxidation reduction pathway across the inner membrane

Yeast is similar to what we have

Question 18

The 2-stage folding funnel

Answer 18

All the different conformations of unfolded proteins is along the top – conformational entropy

Trying to reduce the number of conformations to bring down the conformational entropy

The molten globule state is characteristic of this region – close to the native structure but lots of energy barriers it needs to come across – could have the wrong proline or the wrong disulphide bond – this slows down protein folding

The native structure is the lowest energy state

Question 19

By what pathways does the protein adopt its native conformation?

Answer 19

There are parallel pathways & different ones may be used by the polypeptide under different conditions

The evolutionary aspect of alternative pathways is that if conditions change & dominant routes becomes unfavourable, another apparent pathway can emerge from the transiently populated ensembles to replace it

Proteins with very defined folding pathways are not able to evolve as well as pathways that have alternative pathways

Question 20

How do chaperones help macromolecular crowding?

Answer 20

Chaperones prevent interaction with other molecules when protein is trying to fold

Proteins are packed together into the cell

New proteins secreted into the environment with all the hydrophobic side chains sticking out will just cause the protein to stick to anything

Question 21

Involvement of Hsp70 & Hsp60 in protein folding

Answer 21

Hsp70s look after young polypeptides & allow it form an intermediate or its final state

Sometimes intermediates cannot go into the final folded state but they need Hsp60 like machinery

Late folding will happen but will happen more efficiently in the presence of the correct chaperones

Question 22

Hsp60 in protein folding

Answer 22

Hsp60 proteins deal with the need of some proteins to overcome a transition state not caused by prolines or cysteines

Hsp60 proteins form these environments inside them

• Donut proteins with 7 members & composed of 2 layers of GroEL protein
• Essentially proteins go inside & either by cleavage or ATP or not they can be folded & allowed to adopt the final stable state & then they can be released

Persuades the protein to flip into the correct structure

Question 23

Examples of diseases of protein folding

Answer 23

Alzheimers
Cancer
Mad cow disease
CF