Block B Lecture 2 - Protein Folding and Degradation

Question 1

What does it usually mean if a protein isn’t in it’s one “native” structure?

Answer 1

That the protein isn’t active

(Slide 4)

Question 2

What are 2 ways which a protein can be active, despite it not being in it’s “native” conformation?

Answer 2

By binding of ligands or post-translational modifications (PTMs)

(Slide 4)

Question 3

What form of a protein is more thermodynamically stable, the folded or unfolded forms?

Answer 3

Folded

(Slide 5)

Question 4

How quickly / slowly do proteins fold / unfold?

Answer 4

It starts slowly and then accelerates

(Slide 6)

Question 5

What are 4 forces which drive protein folding?

Answer 5

Hydrophobic forces

Close packing of the protein core

Hydrogen bonding

Salt bridges

(Slide 6)

Question 6

Which force is the most important in protein folding and why?

Answer 6

Hydrophobic forces as hydrophobicity dictates the overall location of amino acids residues in a protein

(Slide 6)

Question 7

Where is rotation in a protein permitted?

Answer 7

About the N-Cα bond (the phi (Φ)) bond and the Cα-carbonyl carbon bond (the psi (Ψ)) bond

(Slide 7)

Question 8

What 2 things make protein folding possible?

Answer 8

Restrictions set by the rigidity of the peptide bond

A restricted set of allowed Φ and Ψ angles (due to steric hindrance)

(Slide 7)

Question 9

What is steric hindrance?

Answer 9

When atoms or groups within a molecule are too close together, leading to repulsion and restricted movement. This sets allowed Φ and Ψ angles

(Slide 7)

Question 10

What is Levinthal’s paradox and what does it suggest?

Answer 10

Protein folding spontaneous, which takes between 10ms - 1 sec.

If a protein of 100 amino acids (assuming 3 conformation of Φ and Ψ angles), that means there are 3^100 possible conformations. Multiply this by the time it takes to fold gives you around 10^27 years.

This means that protein folding cannot be completely random and must be organised in some way

I.e - too many possible conformations for proteins to go through them all - folding must be organised

(Slide 8)

Question 11

What are the 8 steps of the pathways of protein folding?

Answer 11

1. Start with an unfolded chain
2. Short stretches of secondary structures form (such as α-helices and β-strands), which act as nuclei and flicker in and out of existence
3. The nuclei grow by spreading and by adhesion between nuclei, forming extended α-helices and β-strands
4. These secondary structures begin to associate
5. In multidomain proteins the protein reaches a molten globule state, where most secondary structures are correctly formed and the tertiary structure begins development
6. Protein folds into distinct domains
7. The folded domains then condense into a fully folded, functional conformation
8. If applicable, a quaternary structure forms

(Slide 9)

Question 12

What can happen if the protein folding pathway doesn’t work properly?

Answer 12

Proteins can get enter an energetic dead end (a “cul-de-sac”), which can cause aggregates which can cause protein plaques and disease

(Slide 10)

Question 13

Some proteins need assistance to fold properly. What are 3 groups of proteins / enzymes which can help proteins fold?

Answer 13

Folding accessory proteins

Molecular chaperones

Chaperonins

(Slide 15)

Question 14

What are folding accessory proteins?

Answer 14

Enzymes which reorganise certain points in the new-born protein.

Examples include:

protein disulphide isomerases and peptidyl prolyl cis-trans isomerases

(Slide 15)

Question 15

What are molecular chaperones?

Answer 15

They are proteins which bind to proteins as they are synthesised on the ribosomes and prevent them from aggregating, which can prevent proteins from forming undesirable interactions with other proteins

(Slides 15 and 17)

Question 16

What are chaperonins?

Answer 16

They are multi-subunit complexes which help mis-folded proteins to achieve the correct formation

(Slide 15)

Question 17

What does protein disulphide isomerase do and how does it achieve this?

Answer 17

It shuffles the -S-S- bonds until they form between the correct cysteine residues. The enzyme has a groove for binding the substrate and an exposed disulphide on the surface

(Slide 16)

Question 18

What does peptidyl prolyl cis-trans isomerase do and why is this important?

Answer 18

It converts trans proline-X (where X is any amino acids) bonds into cis-bonds, which allows proline to fit into turns

(Slide 16)

Question 19

How can proteins aggregate when being synthesised?

Answer 19

As when they are synthesised, some hydrophobic patches may be initially exposed. If these are left uncovered or unfolded, these hydrophobic patches can stick together resulting in proteins aggregating

(Slide 18)

Question 20

How do molecular chaperones prevent proteins aggregating?

Answer 20

As they have a hydrophobic pocket which binds hydrophobic sections of a newly formed proteins. They bind and release the new-born protein repeatedly, which helps it to fold correctly

Note: This process is driven by ATP
(Slide 18)

Question 21

What is the usual structure of molecular chaperones?

Answer 21

They are usually complexes of several associated proteins

(Slide 18)

Question 22

What type of proteins are many chaperone proteins, and why do scientists believe this is the case?

Answer 22

Heat shock proteins, as their production increases to protect proteins which are in heat shock and other stresses.

Note: chaperones are essential under all conditions, they just appear me in cells in heat shock

(Slide 19)

Question 23

What are “heat shock” proteins?

Answer 23

Proteins which help cells survive environmental stresses like heat, cold, and oxygen deprivation

(Slide 19)

Question 24

What are the steps of the Hsp70 (DnaK) system in E.coli?

Answer 24

1. Hsp40 (DnaJ) delivers the unfolded protein to Hsp70 which binds the unfolded protein in its ATP-bound state
2. Hsp70 hydrolyses its bound ATP (to ADP), which results in the formation of a stable complex as Hsp70 tightens its grip on the protein
3. GrpE binds and promotes ADP release, which allows ATP to bind again. The resulting conformational change in Hsp70 causes it to loosen its grip
4. ATP binding again then triggers the release of the protein. This also resets Hsp70 for the next cycle

(Slide 20)

Question 25

Name an example of a chaperonin which is found in E.coli, another found in eukaryotes and another in chloroplasts.

Answer 25

E.coli: GroEL Eukaryotes: TCiP Chloroplasts: Cpn60 (Slide 21)

Question 26

What is the structure of chaperonin proteins?

Answer 26

They are made up of 14 subunits, with the individual polypeptides being heat shock proteins. They are very large complexes which are barrel-shaped. (Slide 21)

Question 27

What type of proteins require chaperonins to assist with folding?

Answer 27

Particularly different proteins, such as cytoskeletal proteins (Slide 21)

Question 28

How does a protein interact with a chaperonin?

Answer 28

It enters the barrel, where it folds to its native shape Note: sometimes several cycles may be required (Slide 21)

Question 29

What does release of a protein from a chaperonin require?

Answer 29

14 ATP and removal of GroES, which forms a cap over the barrel (Slide 21)

Question 30

What are the steps of chaperonin assisted protein folding?

Answer 30

1. GroEL has 2 stacked rings with each forming a chamber. The partially folded proteins enter this with the hydrophobic interior binding the protein 2. GroES and and ATP binds to GroEL, which causes a conformational change, expanding the chamber. The protein is also encapsulated, preventing aggregation 3. ATP hydrolysis occurs and the chamber undergoes further conformational changes. The protein is forced into a different environment, aiding folding 4. When the protein is correctly folded, it is released from the chamber back into the cytosol. If the protein is still misfolded, another cycle may occur 5. GroES and ADP dissociate, which resets GroEL for another cycle (Slide 22)

Question 31

What are 5 examples of post-translational modifications?

Answer 31

Answers Include: Phosphorylation Glycosylation N-terminal acetylation Hydroxylation of proline Farnesylation Ubiquitinoylation (degradation) Carboxylation of glutamate (in blood clotting factors) (Slide 25)

Question 32

Where does phosphorylation occur and what is it used to regulate?

Answer 32

It occurs on the hydroxyl groups of serine, threonine or tyrosine and is used to regulate the activity of enzymes in metabolism or signalling (Slide 25)

Question 33

What is glycosylation?

Answer 33

Addition of a sugar moiety on an asparagine (N-glycosylation), threonine or tyrosine (O-glycosylation) and it most commonly occurs on extracellular proteins. It protects the proteins and may be involved in recognition (Slide 25)

Question 34

What is N-terminal acetylation and what does it prevent?

Answer 34

it is when an acetyl group is added to the beginning of a protein. It prevents rapid degradation (Slide 25)

Question 35

What are 3 factors which affect protein lifespan?

Answer 35

N-acetylation, protein mis-folding and damage to the protein (Slide 26)

Question 36

Where do non-specific and specific degradation occur?

Answer 36

Non-specific degradation occurs in lysosomes whereas specific degradation occurs in proteasomes (Slide 26)

Question 37

What are proteasomes?

Answer 37

Protein complexes that break down unwanted proteins in cells (Slide 26)

Question 38

What are proteins which are to be degraded tagged with in specific degradation?

Answer 38

A small protein called ubiquitin (Slide 26)

Question 39

What are the 3 steps of ubiquitinoylation?

Answer 39

1. Ubiquitin is linked through it's C-terminal glycine to a cysteine residue of an E1 class enzyme via a thioester bond (a bond between a thiol group (-SH) and an acid (acyl) group) 2. The activated ubiquitin is then transferred to a cysteine residue of an E2 class enzyme 3. Then, E3 class enzymes catalyses the amide linkage (a bond between a carbonyl and an amino group) between the C-terminal glycine of ubiquitin with the amino group of a lysine residue within the target protein. This can occur directly or through an E3-ubiquitin intermediate (Slides 28 - 30)

Question 40

What is ubiquitinoylation affected by?

Answer 40

The N-terminal residue in the target protein (Slide 31)

Question 41

Which N-terminal amino acids cause rapid degradation and which protect against proteolysis concerning ubiquitylation??

Answer 41

Arginine, Lysine, Tyrosine and leucine cause rapid degradation whereas alanine, valine, serine, threonine, glycine and cysteine are stabilising and protect against proteolysis (Slide 31)

Question 42

What happens to proteins which are labelled with ubiquitin?

Answer 42

They are fed into the cells waste disposers, known as the proteosomes. Ubiquitin is recognised as a sort of key in the lock and signals that a protein is on the way to being disassembled (Slide 31)

Question 43

What happens to ubiquitin on labelled proteins?

Answer 43

It is disconnected after the protein reaches the proteosome but prior to the protein being degraded so it can be re-used (Slide 31)

Question 44

How is Alzheimer's disease caused by protein misfolding?

Answer 44

As misfolding causes the amyloid precursor protein and the microtubule-binding protein to accumulate, which then associate into stable filaments, then form tangles and eventually amyloid plaques in the brain which are resistant to degradation (Slide 32)

Question 45

What are tangles?

Answer 45

Abnormal accumulations of proteins which form inside a neuron (Slide 32)

Question 46

How do misfolded proteins cause bovine spongiform encephalopathy (BSE) and Variant Creutzfeldt-Jakob disease (vCJD)?

Answer 46

They are both caused by prions. (Slide 32)

Question 47

What are prions?

Answer 47

Proteins which can be abnormally folded and degraded brain proteins. They have different structures and the conformational change is autocatalytic. The abnormal prion protein (PrP) is partially resistant to proteolysis and accumulates in the brain resulting in aggregation and eventually plaques (Slide 32)

Question 48

What are the structural differences between PrPC (normal prion protein) and PrPSc (abnormal prion protein)?

Answer 48

PrPc is comprised of a lot more α-helices compared to β-sheets (42% compared to 3%) whereas PrPSc is comprised of a lot more β-sheets compared to α-helices (54% compared to 21%) (Slide 33)