Alfonso De Simone Flashcards
General Facts about protein folding - introductory level

What did the Anfinsen experiment show us?
What is the Anfinsen experiment? - Showed that the thermodynamic minimum protein conformation is the native state
When left on its own - the protein will fold in a way to reach this thermodynamic minimum
Outline the steps of the Anfinsen experiment
Procedure
- Denature ribonuclease A (4 disulfide bonds) with 8 M Urea and b-mercaptoethanol to totally unfold the protein in a random coil state having no activity
- Removal of Beta-merceptoenthanol and then urea (attempt to renature protein) –> resulted in protein with no function
Why?
If you were to allow the protein to renature in absence of denaturants we would only get 1% enzymatic activity –> Due to the fact that the probability of one of the Cys to form the correct bond upon renaturation would be 1/7 and then that one of the remaining 6 forming the correct bond would be 1/6, etc. Hence, there is 1/105 chance that all the Cys form the correct bond
- But! Further addition of trace amounts of B-mercaptoethanol converts the scrambled form into native pattern explanation –> This allows non-physiological disulphide bridges to be broken allowing the correct bonds to be made and thus the correct protein structure
Conclusion - This shows us that that the native form correspond to the thermodynamically most stable conformation –> lowest in energy.

In the Anfinsen experiment what other method could be used to reduce the time of renaturation of the scrambled protein?
The time of renaturation of the scrambled protein can be reduced by using protein disulphide isomerase (PDI) which catalyses the disulphide bond interchange.
When is a protein most conformationally stable?

Difference between chemical and conformational stability?

What DG is required for a protein to fold into it’s native conformation? What are some examples of favourable and unfavourable process that occur during protein folding?

What are the external factors, outside the realm of the protein, that influence protein folding?

What are the determinants of protein folding?
Determinants of protein folding –> non-covalent interactions, covalent interactions, compaction, hierarchy, adaptability and sequence versatility.

Why has evolution selected for an average stability around 5 to 10 kcal/mol?
The average stability of a small monomeric protein is only 5 to 10 kcal/mol (after taking into account positive and negative interactions)
Shows us that evolution has resulted in only a marginal stability of the folded state over the unfolded state –> important because it allows cells to ‘remove’/degrade proteins as the folded state is only slightly energetically stable.
Outline the importance of… Covalent interactions, compaction, hierarchy, adaptability and sequence versatility (determinants of protein folding) in protein folding??

How do we determine protein stability?

Definition and causes of denaturation?
Definition Denaturation –> Loss of native structure integrity with accompanying loss of activity.
Causes of denaturation –> heat or cold; pH extremes; organic solvents or chaotropic agents: urea and guanidinium hydrochloride.
How do we plot protein unfolding curve? Are there any important point we should remember?
Plot
Denaturant - X-axis
Percent unfolded - dependent on what is being measured to gauge folding - Y axis

What is Circular Dichroism? What information about protein folding to we obtain using this analytical technique?

What are the three different types of wave oscillations? Background to CD

Why is there differential absorption in protein’s/2o structures?

How is Detla Ɛ (differential absorption calculated) for CD?
Differential absorbance (Delta epsilon) = L.H polarized light - R.H polarized light
Application: Alpha helix - more left handed light absorbed at low wavelengths resulting in positive DƐ , at around 200 there is no difference and after 200 more right handed light absorbed

Does the A.A sequence or 2o structure influence the CD absorption more at low wavelengths?
Different wavelength interaction with the A.A. and secondary structure
- A.A (small scale) more influential at smaller wavelength - A.A chirality has a greater impact on a smaller scale
- Secondary structure is more influential at larger wavelengths
Explains why at extremely large wavelengths there no distinction between 2o structures as the wavelength is too large to interact with structures.
From a measurement perspective…
What happens when equal amounts of L.H polarized and R.H polarized light are absorbed?
What happens when more R.H polarized light is absorbed relative to L.H polarized light?

Pros and Cons of using CD?

What is the Levinthal paradox?
The Levinthal paradox –> States that the folding can not be accomplished by random search/random process of folding there has to be pathway –> if it were to happen by random search - protein folding would take too long to occur too many different conformation combinations.
How is it possible to get proteins to fold in microseconds, if there are so many different possible conformations?

What is the two-state model of folding state?
No stable intermediate species between the native and the unfolded state.





















































