lecture 5 Flashcards
Describe the function of HSP70s in protein folding.
- HSP70s interact with nascent polypeptides
- As the nascent polypeptide emerges from the ribosome, hydrophobic amino acids can aggregate to avoid contacting water
- ATP-bound HP70s bind to short stretches of hydrophobic amino acids exposed on the protein’s surface and hydrolyse ATP to ADP
- HSP70 releases the protein when ATP has been exchanged for ADP
- Most proteins undergo many cycles of ATP hydrolysis before they are correctly folded
- In a cell with no HSP70 present: hydrophobic patches of aas aggregate, trapping the protein in an incorrect conformation or allowing it to aggregate with other proteins in the cell
- In a cell WITH Hsp70 present: stretches of amino acids bind to a peptide-binding groove on Hsp70. Aggregation is prevented.
(in summary)
- Hsp70s are protein-folding chaperones that function early in the protein folding process during translation
- ATP-bound Hsp70s bind to short stretches of hydrophobic amino acids exposed on the protein’s surface, preventing aggregation
- Hsp70s aid correct folding by hydrolysing ATP to ADP
In protein folding, describe the state that is referred to as a ‘molten globule’.
The molten globule state is the state of the protein after it is first fully released from the ribosome.
It is a quite flexible and dynamic state: the protein is partially folded but not fully folded.
It tends to be more open (and obviously less folded) than the fully folded form.
The protein requires help/energy input to reach the its final/lowest energy state from the molten globule form.
Describe the function of an E3 ubiquitin ligase in marking proteins with ubiquitin.
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What is typically the earliest step following translation that is required before a functional protein results?
Correct folding. This occurs through the formation of non-covalent interactions between the amino acids forming secondary and tertiary structures.
There are further modifications that can occur.
When does protein folding begin?
Often as soon is the polypeptide chain grows out of the ribosome. E.g. the first domain of a protein folding before the second has been extruded.
Even when it is released from the ribosome, the protein is not in its fully folded state.
What is the current thinking regarding how proteins get from their ‘molten globule’ state to their correctly folded form?
Current thinking is that proteins progress from the molten globule state to their correctly folded form through a series of steps catalysed by chaperones.
This might happen multiple times before the protein gets to its fully folded state.
If it is unable to reach its final state, the protein will be degraded.
How were heat shock proteins first discovered?
An Italian scientist was working with drosophila and was the first to see that increased expression of heat shock protein genes occurred in drosophila that had been briefly exposed to sub-lethal high temperature.
Later on, after identifying the heat shock proteins, they placed the flies in an oven up to 40ºC and also fed them radioactivity. This was to see the newly formed proteins.
They extracted proteins from flies that had grown at 28ºC and separated the proteins by molecular weight and charge on a gel. They compared this with those exposed to 40ºC. The radioactivity helped display newly formed proteins and they noticed in the 40 flies regular formation of a series of proteins at about the 60-70 kD, corresponding with the heat shock proteins.
What are heat shock proteins?
- many protein folding chaperones are heat-shock proteins
- HSPs are found in all organisms, from bacteria to humans and plants.
- HSP60s and 70s are large protein families, with different family members functioning in different organelles
- BiP is a HSP70 targeted to the endoplasmic reticulum
- HSP60s are sometimes referred to as chaperonins
- HSP70s function in the early stages of the protein-folding process, beginning with translation. (Often when the protein is still being extruded from the ribosome.)
- Many proteins can fold with the aid of HSP70s alone.
- HSP60s act later in the process and provide a central cavity in which protein folding is facilitated.
- Both the HSP70 and HSP60 pathways require energy in the form of ATP hydrolysis.
Which HSP70/60 is particular to the following organisms/elles: bacteria, eukaryotic cytosol, endoplasmic reticulum, mitochondria, chloroplasts, cytosol.
HSP70:
- bacteria: dnaK
- Eukaryotic cytosol: Hsp70
- ER: BiP
- Mitochondria: Grp25
HSP60:
- bacteria: GroEL
- Mitochondria: Hsp60
- Chloroplasts: Rubisco-binding protein
- Cytosol: TRiC
Describe the function of Hsp60s in protein folding.
- Hsp60s form large barrel-shaped structures
- A misfolded protein is initially captured through hydrophobic interactions with the barrel rim
- ATP binding plus a protein cap (GroES) broaden the barrel and stretch the protein
- Protein refolding occurs within the enclosed space
- ATP hydrolysis weakens the complex
- Protein is released following further ATP binding and the cycle repeats if the protein is not correctly folded
- Only half of the symmetrical barrel is occupied at a time
Summarise protein folding
Some proteins correctly fold without help.
Many need the help of a molecular chaperone to get from the molten globular state to get to the fully folded conformation.
Some don’t manage to fold correctly and these are gotten rid of by the cell through the proteasome degradation pathway.
It is very important to prevent the formation of protein aggregates in the cell. This happens when hydrophobic patches are exposed.
What is a proteasome?
- the 26S proteasome is a large, ATP-dependent protease that degrades many of the aberrant proteins produced in the cytoplasm and ER to small peptides
- Makes up about 1% of proteins in the cell (may seem small, but actually represents that it is quite abundant within the cell
- It has a really important role within the cell
- It is made up of segments including a central hollow cavity that contains the protease active sites
- It is important that the active sites are not exposed or else they could go around cleaving any old protein: needs to be regulated
- 19S regulatory molecules on either end act as gates, ensuring only appropriate proteins enter
How do proteasomes work?
- proteins targeted for degradation are tagged with a polyubiquitin chain
- the 26S proteasome degrades polyubiquitinated proteins
- The regulatory particle recognises the polyubiquitin chain and transfers it to the catalytic core
- At an early stage the ubiquitin is removed and recycled
- The ‘gate’ segments also work to unfold the protein as it passes through
- As the protein is fed through a processive enzyme cleaves it up into small polypeptides continuously until the whole protein is degraded
How are proteins targeted for ubiquitinylation?
- ubiquitin is added selectively to proteins by ubiquitin ligases
- the ubiquitin ligase is a complex of several proteins
- the key protein is the F-box protein that is used to select the right protein target for ubiquitinylation
- The E2 conjugating enzyme is that which transfers the ubiquitin to the target protein
- There are many F-box proteins in the cell that target specific proteins for degradation
- They can often recognise hydrophobic exposed regions, targeting those proteins for degradation
Why do cells get rid of proteins?
- misfolded: chucking out garbage
- regulated: start doing stuff e.g. removing a repressor so that a process can proceed, or get rid of a receptor so that it is no longer functional in a particular signalling pathway
- plants have more than 550 F-box proteins: used heavily in regulating processes, not just degrading misfolded proteins
