Protein folding and stability

Question 1

Cystic fibrosis involves misfolding and resulting lack of a protein involved in chloride ion transport across membranes.
Many neurodivergence disorders involve abnormal protein…

Answer 1

Aggregation.

Question 2

List a few of these neurodivergence disorders.

Answer 2

Prion diseases (creutzfeldt-Jakob disease)
Alzheimer’s disease
Parkinson’s disease

Question 3

How are toxic aggregates formed?

Answer 3

Partly folded/misfolded polypeptides or fragments may sometimes associate with similar chains.

Question 4

How are proteins held together?

Answer 4

By an accumulation of weak forces.

Question 5

Why are proteins described as only marginally stable?

Answer 5

They are not that much more stable than their unfolded states.

Question 6

How are proteins normally denatured?

Answer 6

By extremes of pH (most only stable from pH 5-9).
By high temperatures.

Question 7

Proteins are not usually stable in organic solvents.
Why?

Answer 7

This strips essential water.

Question 8

What is protein folding based on?

Answer 8

Primary structure.

Question 9

Protein folding is difficult to accurately predict based on…

Answer 9

Amino acid sequence.

Question 10

What kind of a process is protein folding?

Answer 10

equilibrium

Question 11

How do proteins fold?

Answer 11

To their most energetically favourable conformation and spontaneously.

Question 12

What external factor has a major role in folding proteins?

Answer 12

The solvent.

Question 13

When is a protein’s contact with the solvent maximal?

Answer 13

In unfolded states.

Question 14

List all the forces and interactions that stabilise the 3D structure of proteins.

Answer 14

• hydrophobic interactions
• ionic interactions
• van der waals interactions
• hydrogen bonds
• disulphide bonds

Question 15

Why do proteins tend to be quite stable if they have disulphide bonds?

Answer 15

These are the only covalent interactions.

Question 16

Why are hydrophobic amino acids found inside of globular proteins? Why do pro

Answer 16

They repel water.

Question 17

Hydrophobic amino acids have side chains that are…

Answer 17

non-polar.

Question 18

What kind of core do proteins usually have?

Answer 18

Hydrophobic

Question 19

When do clathrate structures of water molecules form?

Answer 19

During the hydrophobic effect water molecules form a cage like structure when they hydrogen bond with each other but (oil) droplets limit hydrogen bonds.

Question 20

What does protein folding do for hydrophobic residues?

Answer 20

Minimises the contact with water for the hydrophobic residues.

Question 21

What is a salt bridge?

Answer 21

Bonds between oppositely charged residues that are sufficiently close to each other to experience electrostatic attraction.

For instance, an amino acid with a positive side chain can form an ionic bond with an amino acid with a negative side chain.

Question 22

For an ionic bond to form, it requires the removal of a…

Answer 22

solvation shell (where water molecules meet the polypeptide).

Question 24

Why?

Answer 24

To offset (balance) energy liberated (set free) when bond is made.

Question 25

What are van der Waals interactions?

Answer 25

Non-covalent interactions between electrically neutral molecules arising from electrostatic interactions between permanent dipoles and/or instantaneous dipoles and/or induced dipoles.

Question 26

Describe the formation of London dispersion forces (a type of van der Waal interaction).

Answer 26

Chance charge separation creates an instantaneous dipole and a second molecule has a charge separation induced by the first molecule. Positive side of first molecule attracted to negative side of second molecule.

Question 27

How do van der Waals forces compare with ionic interactions within a protein?

Answer 27

Much weaker (but there are many of them within a protein).

Question 28

Interactions have a positive component and a...

Answer 28

negative component.

Question 29

What happens when molecules get too close?

Answer 29

Electron clouds repel.

Question 30

What is the van der Waals distance? (If confused, look at PowerPoint).

Answer 30

The optimal distance between 2 atoms.

Question 31

What is the net force a result of?

Answer 31

The Leonard-Jones potential (energy = A/r^12 - B/r^6). r^-12 = repulsive energy r^-6 = attractive energy

Question 32

How do you find the optimal distance on a graph?

Answer 32

Distance (in angstroms) from 0 to when net energy (in kcal/mol) line reaches lowest energy point on graph.

Question 33

What determines the optimal van der Waals radius?

Answer 33

Attractive and repulsive forces.

Question 34

Order the following van der Waals forces in terms of strength.

Answer 34

interactions between permanent dipoles > dipole-induced dipole interactions > London dispersion forces

Question 35

What can maximise van der Waal interactions between atoms?

Answer 35

optimal packing

Question 36

What are hydrogen bonds?

Answer 36

Electrostatic interactions between a weakly acidic donor, D, group (hydrogen partially letting go of) and an acceptor atom, A, that bears a lone pair of electrons.

Question 37

In a hydrogen bond, the hydrogen atom is sandwiched between...

Answer 37

two electronegative atoms (usually O or N).

Question 38

There are many hydrogen bonds in alpha-helices and beta-sheets. What is the ideal bond length?

Answer 38

2.7 - 3.1 Å

Question 39

How many residues is an alpha helix turn?

Answer 39

3.6

Question 40

What is the pitch distance of an alpha helix (the distance the helix rises along its axis per turn)?

Answer 40

5.4 Å

Question 41

How is hydrogen bonding arranged in a helix to result in the optimal length 2.8 Å?

Answer 41

The peptide C=O group of the nth residue points along the helix towards the peptide N-H group of the (n+4)th residue.

Question 42

Where do the R-groups point in an alpha helix?

Answer 42

outwards

Question 43

Any molecules that can form hydrogen bonds with each other can from hydrogen bonds with...

Answer 43

water molecules.

Question 44

Due to competition with water molecules, the hydrogen bonds formed between two molecules dissolved in water are relatively...

Answer 44

weak.

Question 45

What are disulphide bonds?

Answer 45

Covalent bonds between two cysteine amino acids in the protein chain. Only covalent bond found to hold protein in its correct fold.

Question 46

What kind of proteins are disulphide bonds mainly found in?

Answer 46

Proteins which are exported from the cell and have to be stable at high temperatures.

Question 47

Although some forces may be strong, why do they only make weak contributions to protein stability in the unfolded state?

Answer 47

Similar strength interactions are made with the solvent.

Question 48

When are hydrophobic forces exposed to the solvent and shielded from the solvent?

Answer 48

Exposed = unfolded. Shielded = folded.

Question 49

When are charged groups solvated (interact with solvent) in the protein?

Answer 49

In the proteins unfolded state.

Question 50

What happens to charged groups in the folded state of a protein?

Answer 50

Ionic bonds form within the protein.

Question 51

What do van der Waals forces form between in terms of proteins in their folded and unfolded states?

Answer 51

Unfolded = protein and water. Folded = within protein.

Question 52

What do hydrogen bonds form between in terms of proteins in their folded and unfolded states?

Answer 52

Unfolded = protein and water. Folded = within protein.

Question 53

When are disulphide bonds present in terms of the state of a protein?

Answer 53

Only present in protein's folded state.

Question 54

Who conducted the ribonuclease refolding experiment?

Answer 54

Christian B. Anfinsen

Question 55

What was the ribonuclease refolding experiment?

Answer 55

When beta-mercaptoethanol and urea are added, the ribonuclease is denatured so there is no enzymatic activity. When beta-mercaptoethanol and urea were slowly removed, the protein refolded and correct disulphide bonds reformed giving 100% activity. When only beta-mercaptoethanol was removed, cysteines formed random disulphides and upon subsequent removal of urea approximately 1% of activity is restored.

Question 56

How many cysteines are in ribonuclease?

Answer 56

8

Question 57

Using the question above, where does the 1% come from? (If confused, look at summary sheet).

Answer 57

1/7 x 1/5 x 1/3 x 1/1 = 1/105 ~ 1%

Question 58

Correct protein folding is helped in the cell by proteins called...

Answer 58

chaperonins.

Question 59

Why do chaperonins exist if proteins can fold spontaneously?

Answer 59

To stop proteins aggregating with other unfolded proteins (not to tell proteins how to fold).

Question 60

What is the Levinthal paradox?

Answer 60

Finding the native folded state of a protein by a random search among all possible configurations. This would take longer than the apparent age of the universe.

Question 61

How long does it take for a protein to fold?

Answer 61

Less than or equal to seconds.

Question 62

How do proteins fold to native conformations?

Answer 62

By directed pathways rather than random search methods. The protein forms secondary structure elements to make a core.