UPR recognition Flashcards
Why are proteins sometimes not folded in vivo?
- They fold slowly
- They are missing a vital component
- They are mutated or modified
- They are designed to be unfolded
Why is folding a cooperative event?
Partial folding is relatively unfavourable energetically. This means even small chemical modifications can lead a protein to become unstable. The free energy difference between the forces acting to keep a protein folded an those acting to stabilise an unfolded protein is tiny: noncovalent interactions are weak. Tipping the balance can be easy.
Is the alternative to folding always unfolded?
This is not easy to estimate. Modern technology including NMR relaxation methods describe unfolded states as favouring some orientations and these can be locally folded-like or “misfolded”-like (i.e. structured in a non-native way). Typically, when a protein is liberated into the solvent by a ribosome or chaperone, our current knowledge suggests that the first thing to happen is HYDROPHOBIC COLLAPSE. Alternative conformations that have been described and are known to have some stability have properties of a MOLTEN GLOBULE. As a whole, these statements suggest a highly dynamic structure but one in which the protein hides as much of its hydrophobic (hydrocarbon) chains as possible. Some proteins are likely to be more successful at this than others but an inability to fold correctly is likely to be reflected by exposure of hydrophobic groups and increased chain flexibility.
What interactions are present within a protein?
- Van der Waals packing (rather specific?)
- hydrogen bonding networks (backbone ones are not very specific)
- a small number of salt bridges(specific)
- Hydrophobic interactions (non-specific in initial highly unfolded states).
What do proteins have responsible for non specific binding have?
Hydrophobic surfaces are the only non-specific property of an unfolded state that is not shared by folded states. This idea has dominated the field and indeed, all proteins responsible for binding non-specifically to any unfolded protein have a protein binding site that is rich in hydrophobic residues.
How many evidences are there for chaperones being responsible for UPR recognition?
Three
What is evidence 1 for chaperones and UPR recognition?
Binding of GroEL to unfolded lactate dehydrogenase (Badcoe et al). Figures a-e represent recovery of enzyme activity as a function of time in the absence and presence of GroEL. Without ATP, the protein remains bound and folding is stopped. The less folded the enzyme, the more it is able to bind. The molten globule does not bind.
What is evidence 2 for chaperones and UPR recognition?
The folded state of the protein actually bound to GroEL is monitored using hydrogen-exchange as a labelling technique.
What is hydrogen labelling?
A protein with a particular structure has an –NH group for each of its amino acids. This hydrogen is particularly labile and will dissociate occasionally to bind the oxygen of water. At the same time, a hydrogen atom can dissociate from the oxygen of water and bind the backbone nitrogen. This is called hydrogen exchange. Hydrogens on carbon atoms do not dissociate at a detectable rate. If the solvent surrounding a protein is swapped to D2O (heavy water) rather than H2O, then exchange will occur, but at different rates for different parts of the molecule. NH groups involved in hydrogen-bonding (for example those involved in secondary structure) or NH groups buried in a tightly packed structure will be protected from exchange compared with surface NH groups. Mapping the position of these different groups (requires NMR) or estimating the degree of labelling by mass spectrometry provides a clue as to how structured a protein is and can potentially map out secondary structure.
What is evidence 3 for chaperones and UPR recognition?
Phage display: some bacteriophage display random 8 residue sequences from the N-terminal of the adsorption protein PIII. By creating a library of N-terminals from sequences found in the yeast genome, binding to Hsp 70 (BiP) can be selected for using Hsp70 immobilised on a plate.
Do UPR receptors sense unfolded proteins directly?
The literature is divided. Model 1 IRE-1 and PERK bind directly to unfolded proteins. Model 2. BiP binds IRE-1, PERK (and ATF6). When [unfolded protein] increases, free [BiP] decreases causing BiP to dissociate. This induces dimerisation of IRE-1 and PERK whereas it releases ATF6 from the ER and allows it to migrate to the Golgi (where it is proteolysed).
How many models are there for UPR arms recognising UPR?
Three
What is model one for UPR arms and UPR recognition?
Binding to IRE-1 in yeast. The flexibility of unfolded proteins allows them to insert extended unstructured segments into the binding sites of IRE-1 and PERK, a little like peptides are seen to bind equally non-specific substrate binding sites of MHC1 or Hsp70. Dimerisation is required for the binding site to be formed.
What is model two for UPR arms and UPR recognition?
IRE-1 (mammalian) cannot bind. The binding site identified by the Walter group is too small in the mammalian protein and is blocked by large bulky side chains. Dimerisation can happen in the absence of unfolded protein substrates: dimerisation is only prevented by the binding of Hsp70 (BiP). The residues that promote dimerisation are conserved.
What is model three for UPR arms and UPR recognition?
Both BiP and direct binding contribute to UPR recognition?
