(L6) Protein folding and chaperones

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What are the characteristics of a folded protein
Compact structure, presence of secondary structures (a-helix and B-pleated sheets), Hydrophobic amino acids non-polar and hydrophilic amino acids, polar.
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Name some problems with protein folding
Formation of incomplete structures, exposed hydrophobic ends can be "sticky" and cause aggregation, Protein folding is not completely spontaneous, sometimes need help from other proteins called chaperones
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What do chaperones do?
Bind to partially folded proteins and catalyse the refolding of misfiled proteins and inhibit protein aggregation
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Which organisms need more chaperone proteins?
Thermophyllic organisms
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What happens to irretrievable misfiled proteins?
They are degraded by proteasomes
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Name some types of chaperones
Hsp90 family (90kDa polypeptides), Hsp70 family (70kDa polypeptides) and Hsp60 family and chaperonins (60kDa polypeptides)
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What does each family have?
An associated co-chaperone
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What are HSP's?
Heat shock proteins, they are produced by the cell in response to heat shock, amounts of chaperons increase greatly when there is an increase in heat (chaperones protect against thermal denaturing)
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Hsp70 family
Bind to exposed stretches of hydrophobic amino acids as polypeptides are synthesised, ATP hydrolysis occurs in both bonding and separation
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Hsp60 family
Found in bacteria, mitochondria and plastids, bind to polypeptides after synthesis is complete and provide a space for protein folding, they mainly stick to proteins <60kDa
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Describe the chaperone in E.coli
Chaperone: GroEL, Hydrophobic regions on incorrectly folded protein bind to GroEL, GroES (Hsp10) a co-chaperone "cap" and ATP bind, protein refolds, ATP hydrolysed, GroES disassociates, releasing refolded protein
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Chaperonins
Hsp60 are members of a wider class of chaperonins, required the folding of ~10% of proteins, they have a cavity for protein folding
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Describe the chaperonin for eukaryotic cytosol
TRiC/CCT, has a built in lid, required for the folding of actin, tubulin, G1/s cyclin (among others), can help multi-domain proteins to fold
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Hsp90 family
Helps to complete folding of some proteins including steroid proteins, including steroid hormone receptors, partially folded 'client' proteins can pass from Hsp70 to Hsp90
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Name a problem with protein targeting
They have complex structures so transport and labelling is an issue
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Name 3 types of transport
1) Gated, through nuclear pores, 2) Tans-membrane, into ER, plastids and mitochondria, 3) Vesicular, membrane bound vesicles
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What are protein "address labels"?
Sequences of amino acids within the protein that specify its destination (signal sequences)
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Where can these 'address labels' be found?
At the amino acid terminal, in the middle, at the carboxy terminus
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What happens to the signal sequence?
It is removed either when that protein arrives at its destination or before
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Give 2 uses of the ER in gene expression
Synthesis of membrane proteins, first stage of movement of proteins to Golgi apparatus and beyond
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Where is the signal sequence for transport to the ER usually found?
Amino acid terminal (signal peptide)
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What is the average length of signal peptides in eukaryotes?
23 amino acids
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What is the usual sequence?
There isn't one apart from usually having about 10 hydrophobic amino acids in the middle
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What do signal peptides bind to?
Signal recognition particle , which directs it to the ER
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What happens to the signal sequence once it reaches the ER?
It binds to the ER while the protein is being synthesised, resulting in rough ER with membrane bound ribosomes
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What is the first step of the mechanism of transport?
mRNA, with signal peptide, binds to ribosome subunits in cytosol, the newly-synthesised signal peptide binds to a signal-recognition particle in the cytosol
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What is the signal recognition particle?
6 polypeptides and 7SL RNA
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What happens once the SRP is bound to the ribosome?
SRP-ribosome complex binds to SRP receptor in the ER membrane, signal peptide is inserted into a translator (ER pore) and folds back through the membrane, the growing peptide threads through the translator making the loop bigger
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Once the protein is synthesised, what happens?
The signal peptide is cleaved and the mature protein is released into the ER lumen and the ribosome subunits, SRP & SRP receptor are displaced and recycled
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What is the difference with the translocation of membrane proteins?
They remain in the membrane (signal peptide not cleaved) and it may thread through it more than once
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Give some characteristics of membrane proteins
They have internal signal sequences (start-transfer sequences), stop-transfer sequences control the formations of loops, proteins are released from translator into the membrane
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How many start-transfer and stop-transfer sequences do membrane proteins have?
It depends, however they have multiple if the protein crosses the membrane several times
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Give an example of a membrane protein
Rhodopsin (in the retina)
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What happens to most proteins that enter the ER?
They get glycosylated
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What does the oligosaccharide contain?
14 sugars: (3 Glucose, 9 Manose, 2 N-acetylglucosamine) OR (3 Glucose, 9 Manose, 2 GlcNAc)
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When the oligosaccharide is synthesised, what is it attached to?
Dolichol phosphate, which spans the membrane
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What is dolichol phosphate?
Long-chain alcohol with 100 carbon atoms
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How does the oligosaccharide, with the dolichol phosphate attached, get attached to a protein?
Membrane-bound oligosaccharyl transferase attached oligosaccharide to asparagine (amino acid) before protein synthesis is complete, via an N-link
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Card 2

Front

Name some problems with protein folding

Back

Formation of incomplete structures, exposed hydrophobic ends can be "sticky" and cause aggregation, Protein folding is not completely spontaneous, sometimes need help from other proteins called chaperones

Card 3

Front

What do chaperones do?

Back

Preview of the front of card 3

Card 4

Front

Which organisms need more chaperone proteins?

Back

Preview of the front of card 4

Card 5

Front

What happens to irretrievable misfiled proteins?

Back

Preview of the front of card 5
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