Protein folding Flashcards Preview

BS2091 Biochemistry I: from genes to proteins > Protein folding > Flashcards

Flashcards in Protein folding Deck (32):

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

Unfolded -> intermediate -> folded


Why do proteins fold?

There is equilibrium between folded and unfolded states
[Anfinsen's experiments]


What is the equation for Kfolding?

Kfolding = [folded] / [unfolded]


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

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


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

delta G = delta H - TdeltaS
Need to consider both enthalpy (H) and entropy (S)


What is delta H?

The change in enthalpy


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

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.


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

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.


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

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


What is entropy, S?

Proportional to logarithm of number of states


How many conformations can an unfolded protein have?

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


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

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)


What is unfavourable about an unfolded protein?

Each hydrophobic side chain reduces the freedom of two water molecules


What is favourable about a folded protein?

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


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

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


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

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.


Is folding reversible?



What did Anfinsen's experiments show?

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


Describe the thermodynamics hypothesis

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


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

Seems that bot can occur


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

Secondary structure elements


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

Compact regions


What happens when concentrated solutions of proteins are heated?

They aggregate


Why do high concentrations of protein aggregate?

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


When are proteins especially prone to aggregation?

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


What molecules are significant in preventing proteins from aggregating?

Chaperone protein


What is the role of chaperone proteins?

Assist folding by preventing aggregation


How do chaperone proteins carry out their function?

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.


Give an example of a chaperone protein



How do the GroEL/GroES chaperone proteins work?

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


Where are the hydrophobic side chains in GroEL?

Hydrophobic side chains face inner chamber


Where are the hydrophobic side chains in GroEL-GroES?

Hydrophobic side chains pulled away from inner chamber