Protein Folding

Question 1

What is proteostasis?

Answer 1

Dynamic equilibrium where protein synthesis and folding is balanced with degradation.

Question 2

What is proteostasis influenced by?

Answer 2

Heat shock response
Unfolded protein response
Aging
Organelles
Inflammation
Transcription/ translation/ foldase/ unfoldase/
protease

Question 3

How common are polymorphisms?

Answer 3

~2 coding polymorphisms per sequence, leading to 18% proteins with a mutation

Question 4

What do molecular chaperones facilitate?

Answer 4

Disulfide bridges
Ionic interactions
H-bonding
Hydrophobic effect

Question 5

What functions do Hsp70, Hsp60, Hsp40 and Hsp100s have

Answer 5

Hsp70 - Holdase: binds short hydrophobic stretches of helical protein
Hsp60- Used for proteins with slow folding to prevent them aggregating.
Hsp40- Co-chaperone that allow ATP hydrolysis that give affinity for the unfolded protein
Hsp100s: disaggregases, usually combined with proteolysis

Question 6

How do disulfide bonds stabilise proteins?

Answer 6

Covalent bond formation makes almost irreversible tertiary contact
Depends on Cys
Difficult to reverse
Can give thermal stability

Question 7

How do ionic bonds stabilise proteins?

Answer 7

Electrostatic interaction between +ve and -ve
Strong and specific as sidechains only have one charge
Interact with solvent via water shields effect

Question 8

How do H-bonds stabilise proteins?

Answer 8

Amide and carbonyl backbones stabilise structure
Interact with water
Present acceptor or donor at every residue but Pro

Question 9

How does hydrophobic effect stabilise proteins?

Answer 9

Water is excluded from non-polar surfaces, maximising H2O H-bonding
Poorly defined geometry as many sidechains interact
When hydrophobic residues get away from water, hydrophobic collapse takes place.

Question 10

What is Hsp70 function?

Answer 10

After heat shock, they bind hydrophobic streches of 7aa
Hsp70 binding sites every 35-40 residues
Prevents hydrophobic interactions and aggregation
2 co-chaperones allow it to translocate in mitochondrial import

Question 11

What is Hsp40 function?

Answer 11

Co-chaperone that helps Hsp70 translocate in mitochondrial import
ATP hydrolysis for affinity for unfolded protein
Nuclear exchange factors (NEF) allow protein release

Question 12

What is Hsp104 function?

Answer 12

Involved in reverse aggregation
ATPase involved in supressing prion Psi
Hexameric ring.
Each monomer contains 2 nucleotide binding domains
polypeptides forced through central channel of the ring, unfolding polypeptide

Question 13

What is Hsp60 (GroEL) function?

Answer 13

Used for proteins that fold slowly to stop them aggregating
Dimer of heptamers
2 stacked proteins which trap the protein and allow it to fold
Affinity for unfolded protein modulated by nucleotide binding
Full activity requires Hsp10 (GroES)

Question 14

Describe how Hsp60/GroEL prevent a protein aggregating while it folds

Answer 14

Groel ring binds GroES in the presence of a nucleotide. This forms a cap with hydrophilic walls, allowing protein to fold without aggregating

Question 15

What is Hsp90 function?

Answer 15

Upregulated by heat shock and binding affinity regulated by ATP
Induces nuclear import of kinases and transcription factors that have not received a signal. This is because they may not have a stable strucutre

Question 16

What example of Hsp90 is there?

Answer 16

Oestrogen receptor cannot fold without a hormone, so forms a stable complex with Hsp90 until the hormone binds.
This then moves to the nucleus and acts as a transcription factor

Question 17

Why might a protein aggregate?

Answer 17

Folding processes, e.g Pro isomerisation and disulfide bond formation are slow

Incomplete domains on partially synthesised proteins may not fold into stable structures on the ribosome

No post-translational modifications

Non-protein binding proteins

High protein concentration making non-specific, intermolecular interactions

The same prion polymorphs are around

Question 18

Why are proline isomerases needed?

Answer 18

Resonance characteristics of Pro do not favour trans as there is no proton on N terminus.
Interconversion of Pro from cis -> trans is 0.05s-1 which slows protein folding.
60% proteins have X-pro isomer

Question 19

What are FK binding proteins?

Answer 19

Target of ascomycin
Peptidyl proline isomerase that can increase folding 300-fold.

Question 20

What are protein disulfide isomerases?

Answer 20

Assist in rearranging disulfide bonds in eukaryotic ER
Oxidation and reduction for random disulfide exhange from DsbA to protein
CXXC motif in the centre of a hydrophobic surface and a Cys with a pKa 6.7 starts the reaction
DsbA is reduced by DsbB which is oxidised by ubiquinol which is oxidised by cytochrome oxidase
DscC is reduced by DsbD and rearranges disulfide bonds via 2 Cysteines

Question 21

What is mitochondrial malate dehyrogenase (MMDH)?