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 is spontaneous, which takes between 10ms - 1 sec.

In 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

How do proteins overcome Levinthal’s paradox?

Answer 11

By following organized pathways involving the formation of intermediate structures and being guided by physical constraints like restricted phi and psi angles.

(Slide 8)

Question 12

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

Answer 12

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 13

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

Answer 13

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

(Slide 10)

Question 14

What are folding accessory proteins?

Answer 14

Proteins that help other proteins fold correctly into their functional 3D shapes. They can recognise certain bonds in the new-born protein and assist in the proper formation of these bonds. They work by preventing aggregation, catalyzing slow folding steps and guiding the protein through intermediate states to ensure the correct final conformation.

(Slide 15)

Question 15

What are 2 examples of classes of folding accessory proteins?

Answer 15

Molecular chaperones

Chaperonins

(Slide 15)

Question 16

What are 2 examples of folding accessory proteins which aren’t molecular chaperones or chaperonins?

Answer 16

Protein disulphide isomerases (PDIs) and peptidyl prolyl cis-trans isomerases (PPIases)

Question 17

What are molecular chaperones?

Answer 17

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 18

What are chaperonins?

Answer 18

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

(Slide 15)

Question 19

What do protein disulphide isomerases (PDIs) do and how does it achieve this?

Answer 19

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 20

What do peptidyl prolyl cis-trans isomerases (PPIases) do and why is this important?

Answer 20

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 21

How can proteins aggregate when being synthesised?

Answer 21

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 22

How do molecular chaperones prevent proteins aggregating?

Answer 22

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 23

What is the usual structure of molecular chaperones?

Answer 23

They are usually complexes of several associated proteins

(Slide 18)

Question 24

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

Answer 24

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 more in cells in heat shock

(Slide 19)

Question 25

What are "heat shock" proteins?

Answer 25

Proteins which help cells survive environmental stresses like heat, cold, and oxygen deprivation (Slide 19)

Question 26

What is the Hsp70 system in E.coli?

Answer 26

It is a system involving Hsp70 molecular chaperone which helps proteins fold into their correct conformation. (Slide 20)

Question 27

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

Answer 27

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. This helps the substrate protein in its folding process by providing the necessary mechanical force. 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 Note: Several cycles of this may be required to get a fully correctly folded protein (Slide 20)

Question 28

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

Answer 28

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

Question 29

What is the structure of chaperonin proteins?

Answer 29

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 30

What type of proteins require chaperonins to assist with folding?

Answer 30

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

Question 31

How does a protein interact with a chaperonin?

Answer 31

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

Question 32

What does release of a protein from a chaperonin require?

Answer 32

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

Question 33

What are the steps of chaperonin assisted protein folding? Use GroEL as an example.

Answer 33

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 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 34

What are 5 examples of post-translational modifications?

Answer 34

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

Question 35

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

Answer 35

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 36

What is glycosylation?

Answer 36

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 37

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

Answer 37

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

Question 38

What are 3 factors which affect protein lifespan?

Answer 38

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

Question 39

Where do non-specific and specific degradation occur?

Answer 39

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

Question 40

What are proteasomes?

Answer 40

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

Question 41

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

Answer 41

A small protein called ubiquitin (Slide 26)

Question 42

What are the 3 steps of ubiquitinoylation?

Answer 42

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 the carbonyl carbon of a carboxyl group (C=O)) 2. The activated ubiquitin is then transferred from the E1 enzyme to a cysteine residue of an E2 class enzyme 3. Then an E3 class enzyme catalyzes the formation of an isopeptide bond (an amide bond between the carbonyl group of the ubiquitin's C-terminal glycine and the amino group of a lysine residue in the target protein). This step attaches ubiquitin to the tagrte protein and can occur either directly or via an E3-ubiquitin intermediate. (Slides 28 - 30)

Question 43

What is ubiquitinoylation affected by?

Answer 43

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

Question 44

What are 3 N-terminal amino acids which cause rapid degradation in ubiquitylation?

Answer 44

Answers Include: Arginine Lysine Tyrosine Leucine (Slide 31)

Question 45

What are 4 N-terminal amino acids which protect against proteolysis in ubiquitylation?

Answer 45

Answers Include: Alanine Valine Serine Threonine Glycine Cystine Methionine (Slide 31)

Question 46

What happens to proteins which are labelled with ubiquitin?

Answer 46

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 47

What happens to ubiquitin on labelled proteins?

Answer 47

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 48

How is Alzheimer's disease caused by protein misfolding?

Answer 48

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 and can interfere with neuronal signalling and cause inflammation. (Slide 32)

Question 49

What are tangles?

Answer 49

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

Question 50

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

Answer 50

Prions become misfolded and aggregate in the brain (Slide 32)

Question 51

What are prions?

Answer 51

Prions are proteins which can misfold into an abnormal form (PrPSc) which resists proteolysis and aggregates in the brain. The conformational change from the normal protein (PrPc) to PrPSc is autocatalytic, meaning it can induce other normal prion proteins to misfold. Aggregation of PrPSc eventually forms plaques, which can contribute to neurodegeneration. (Slide 32)

Question 52

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

Answer 52

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)