L6 - Late stages in protein folding Flashcards
What is the molten globule?
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.)
What separates the early and late stages of protein folding?
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
How do we study the molten globule?
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
What do experiments from creating a stable ‘molten globule state’ under mildly denaturing conditions show?
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
Detecting the molten globule intermediates using fluorescence
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
Why does ANS not bind to unfolded or folded states?
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
Detecting the molten globule intermediates using circular dichroism
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
What does the far UV & the near UV show in CD?
Far UV – secondary structure
Near UV – side chains
Signature state of a native (folded) protein
- Far UV
- Near UV
- Strong Trp fluorescence
- No ANS fluorescence
- Cooperative unfolding
Signature state of a MG protein
- Far UV
- No near UV
- Strong Trp fluorescence
- Strong ANS fluorescence
- No cooperative unfolding
Signature state of an unfolded protein
- No far UV
- No near UV
- No Trp fluorescence
- No ANS fluorescence
- No cooperative unfolding
Reason for slower folding of intermediates
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
Proline isomerisation
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
Peptidyl-prolyl-isomerase (PPI)
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)
Disulphide bridges
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)
Disulphide bridges in E. coli
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
Disulphide bridges in yeast
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
The 2-stage folding funnel
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
By what pathways does the protein adopt its native conformation?
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
How do chaperones help macromolecular crowding?
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
Involvement of Hsp70 & Hsp60 in protein folding
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
Hsp60 in protein folding
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
Examples of diseases of protein folding
Alzheimers
Cancer
Mad cow disease
CF
