Protein Folding

Question 1

Describe the experiment done by Andinsen in 1961 and his key conclusions from this

Answer 1

Experiment: can unfold RNAase with a denaturing reagent (urea-) and a reducing agent (B-mercaptoethanol) breaks the disulphide bonds
If these conditions are taken away and an oxidising agent is present, can refold the protein.
Conclusions: Native structure is the thermodynamically most stable (favoured) state for most proteins.
Native tertiary structure is determined by the primary structure (amino acid sequence) of a protein.

Question 2

What is Levinthal’s paradox?

Answer 2

Protein cannot fold by trying every conformation as there are too many possible conformations for a protein. Folding must occur via intermediates in one or several defined pathways.
Folding funnels- specifically defined folding pathways.

Question 3

How can protein folding be measured?

Answer 3

Can measure the process of unfolding to understand the protein folding sequence. Can measure the the change in energy between the folded state and unfolded state. The transition between states must be in equilibrium to measure this. Can disturb the equilibrium by using urea (best) or temperature- need to be careful with temperature as this can sometimes cause irreversible change.

Question 4

What are the three proposed models for how folding occurs?

Answer 4

The Framework model- protein is unfolded, searches for 2ndry structure, folds and then folds into tertiary structure
The classical nucleation model- proposes there is a single starting nucleation point (e.g. helix or strand) and then the rest of protein folds around this
Hydrophobic collapse- hydrophobic residues cluster into the centre and with this the secondary and tertiary structures are formed around it

Question 5

What is the proposed molten globule?

Answer 5

Partially folded protein which has some components of a folded protein such as secondary structure (e.g. a-helix) and some aspects of an unfolded protein (no tertiary structure)- are flexible so hard to apply standard techniques to visualise them- demonstrate an equilibrium state

Question 6

What evidence is there for no intermediate states and what model was proposed for this?

Answer 6

Chymotrypsin Inhibitor 2 was shown to have no stable intermediates in an experiment in 1991; led to the proposition of the nucleation/condensation model which proposes that the protein foldes around a nucleation point

Question 7

Describe the concept of energy landscapes

Answer 7

Proposed that protein folding is biased due to preferences of amino acids and this causes a protein landscape- native state has the lowest minima but there are other energy states in which the protein can fall with different kinetics- can occupy some pathways longer than others

Question 8

How does unfolding by denaturants (e.g. urea) work?

Answer 8

Two models for this: preferential binding model and preferential solvation model. This states that denaturants such as urea will increase the solubility of both polar and non-polar sidechains and the protein backbone, stabilising the denatured state more than the native state

Question 9

How can an unfolded state be determined in vivo and in vitro?

Answer 9

Can use a fluorescent probe which attaches to molecules such as tryptophan and tyrosine. These molecules are usually in the core of the protein as they are hydrophobic. If the protein is denatured, these will be exposed and so will be detected.
In vitro: can use a solution in a cuvette
In vivo: use a microscope

Question 10

Describe the stages in the rapid mixing and stopped flow technique to determine protein intermediates

Answer 10

1. Have two solutions, one with protein in cell A and one with solution in cell B; put these under high pneumatic drive and high efficiency mixer
2. Pass through a measurement flow cell into a syringe
3. Just prior to stopping, a steady state flow is achieved
4. As solution fills the syringe, the plunger hits a block and stops the flow
5. Kinetics of reaction can be measured
6. Can combine this with detection methods

Question 11

What is psi value analysis?

Answer 11

Measure the equilibria between a native state protein and its unfolded state
Mutate some of the residues inside the protein
Measure the folding and unfolding of mutant compared to the normal pathway
Can compare to identify specific residues essential for folding and unfolding- can also measure energy change (delta G) to determine energy state changes