Protein folding

Question 1

In the simplest way, outline the steps to get to a folded protein

Answer 1

Unfolded -> intermediate -> folded

Question 2

Why do proteins fold?

Answer 2

There is equilibrium between folded and unfolded states

[Anfinsen’s experiments]

Question 3

What is the equation for Kfolding?

Answer 3

Kfolding = [folded] / [unfolded]

Question 4

What is the equation for K and what does this mean?

Answer 4

K = e^(delta G / RT)
Equilibrium is determined by the relative energies of the states, energy of folded state must be lower than unfolded state

Question 5

What is the equation for delta G (Gibb’s free energy) and why do we need to use it?

Answer 5

delta G = delta H - TdeltaS

Need to consider both enthalpy (H) and entropy (S)

Question 6

What is delta H?

Answer 6

The change in enthalpy

Question 7

Describe the enthalpy of hydrogen bonds and how the bonds contribute to folding

Answer 7

The enthalpy of hydrogen bonds - each H-bond is worth ~10-15 kJ/mol
There are many of these in a folded protein.
But in folding the hydrogen bonds between the water molecules and the protein are lost.
There may be more lost than gained. Although the geometry may be better in the protein.

Question 8

Describe the enthalpy of and how the ion pairs (salt bridges) contribute to folding

Answer 8

Very strong (in a vacuum!) but the shielding effect of water reduces this significantly - estimated ~6-20 kJ/mol
Significant energy penalty in removing from aqueous environment means completely buried away from solvent (and therefore very strong) ion pairs are rare.

Question 9

Describe the enthalpy of van der Waals interactions and how the bonds contribute to folding

Answer 9

These are significantly weaker - ~1 kJ/mol
But there are many of them when hydrophobic regions packed in protein interior
So enthalpy makes a contribution to stabilising folded state, ~3 kJ/mol/residue, so for 50 residue protein say 150 kJ/mol

Question 10

What is entropy, S?

Answer 10

Proportional to logarithm of number of states

Question 11

How many conformations can an unfolded protein have?

Answer 11

i) Backbone assume 3 rotational states
ii) Side chain assume 2 rotational states
So 3*2 states per residue
For N residues 6^N

For 50 residue protein at 300K
TdeltaS ~223 kJ/mol
This is much greater than expected enthalpic gain of ~150 kJ/mol

Question 12

Describe the difference in how a water molecule acts in bulk water compared with when a nonpolar group is present

Answer 12

In bulk water:
Water molecule; in bulk water, this water can tumble in all directions; (this water molecule can rotate in many directions without losing hydrogen-bonding partners)

When a nonpolar group is present:
Nonpolar group; orientation of water molecule is restricted; water molecule avoids placing hydrogen-binding groups in this region; (hydrophobic group cannot form hydrogen bonds)

Question 13

What is unfavourable about an unfolded protein?

Answer 13

Each hydrophobic side chain reduces the freedom of two water molecules

Question 14

What is favourable about a folded protein?

Answer 14

Folded protein has all the hydrophobic groups inside, away from water

Question 15

Using an example, show the mathematics of why a folded protein is more favourable than an unfolded protein

Answer 15

For a 50 residue protein, assume half of the residues (25) are hydrophobic, and when unfolded each hydrophobic side chain reduces the freedom of 2 water molecules.
Water molecules have 6 (low energy) states (3 rotational to make 4 interactions).
Estimated entropy gain TdeltaS is ~137 kJ/mol
Protein: deltaG = deltaH - TdeltaS = +73 kJ/mol
Water: deltaG = deltaH - TdeltaS = -137 kJ/mol
Net gain (-) ~64 kJ/mol
PROTEIN FOLDING IS DRIVEN BY AN INCREASE IN WATER ENTROPY

Question 16

How do proteins fold (how do they fold properly)?

Answer 16

Levinthal’s paradox
If an 100 amino acid residue protein has 3 conformations allowed per residue, then there are 3^100 = 5*10^17 seconds, so even femtosecond (10^-15s) sampling would not allow all of the conformations to be sampled.
Small proteins can fold in milliseconds.
So folding must be sequential rather than random process.

Question 17

Is folding reversible?

Answer 17

Often

Question 18

What did Anfinsen’s experiments show?

Answer 18

That all the information required to fold a protein is in its sequence (i.e. the chemical properties of the molecule)

Question 19

Describe the thermodynamics hypothesis

Answer 19

There are 2 possible sequences of events

a) Diffusion collision - secondary structure elements form first, these collide with each other to form the native structure
b) Nucleation condensation - side chains interactions stabilise a compact structure then secondary structure nucleation occurs

Question 20

Which of the two possible sequences of events of the thermodynamics hypothesis is more likely to occur?

Answer 20

Seems that bot can occur

Question 21

What do alpha-helical proteins tend to form first when folding?

Answer 21

Secondary structure elements

Question 22

What do beta-sheet proteins tend to form first when folding?

Answer 22

Compact regions

Question 23

What happens when concentrated solutions of proteins are heated?

Answer 23

They aggregate

Question 24

Why do high concentrations of protein aggregate?

Answer 24

Consider that at low concentrations proteins can fold before encountering another. An unfolded protein will be ‘sticky’ - it will have exposed hydrophobic regions. If the concentration is high, the molecules will stick to each other.
In the cell the concentrations of proteins is very high ~300-400 mg/ml

Question 25

When are proteins especially prone to aggregation?

Answer 25

Proteins as they are being synthesised on ribosomes
Proteins being translocated across membranes
Proteins in cells subject to heat shock

Question 26

What molecules are significant in preventing proteins from aggregating?

Answer 26

Chaperone protein

Question 27

What is the role of chaperone proteins?

Answer 27

Assist folding by preventing aggregation

Question 28

How do chaperone proteins carry out their function?

Answer 28

Bind reversibly to unfolded or partially folded protein through hydrophobic surfaces. Once the protein has folded it will have a hydrophobic surface and lower affinity for the chaperone.

Question 29

Give an example of a chaperone protein

Answer 29

GroEL/Hsp70

Cycle

Question 30

How do the GroEL/GroES chaperone proteins work?

Answer 30

Energy from ATP hydrolysis is used for conformational change so that the inside of the chamber changes from hydrophobic to hydrophilic.
The chambers are known as Anfinsen cages

Question 31

Where are the hydrophobic side chains in GroEL?

Answer 31

Hydrophobic side chains face inner chamber

Question 32

Where are the hydrophobic side chains in GroEL-GroES?

Answer 32

Hydrophobic side chains pulled away from inner chamber