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Proteins - Protein denaturation, protein stability

Q: What happens to a protein after heating?

It can go back to the native state Or it goes back to an unnative state (denatured)

Q: What happens with proteins during spray drying?

The denaturation temperature increases with decreasing water activity, consequently, unfolding does not necessarily take place during spray drying.

Q: What is the effect of pH on proteins?

Gives reversible unfolding. Proteins are more stalbe at ther IEP extreem pH lead to unfolding

Q: What happens to the protein at a very alkaline pH?

hydrolysis of peptide bonds deamidation of Asn and Gln S-S reshuffling destruction of SH Because of this there is more unfolding at high ph then at low ph

Q: Why is a protein most stable at the IEP?

There is the same amount of + as - charge (THERE STILL IS CHARGE!) These can form a interaction and stabilize a protein. If you go far away form the IEP you will have mostly negative or positive charge that repels each other and denatures your protein.

Q: What is the effect of unfolding of detergents?

The detergents bind to an unfolded protein and changes there equilibrium. This makes that the protein prefers to stay in the denatured state. This already happens at 3-8 mN.

Q: What is the effect of organic solvents on protein unfolding?

They weaken the hydrophobic interactions Enhance the formation of peptid H-bonds (therefore increases the strength of the secondary structure) Enhance electrostatic interactions: attraction/repulsion. However if the concentration becomes to high denaturation occurs.

Q: What is the effect or organic solutes (sugars) on protein unfolding?

Solutes formation of H-bonds whit water molecules This results in 'destructuring' of water This results in solubilisation of hydrophobic AA residues This results in unfolding

Q: What is the effect of an unfolded protein?

Can lead to: loss of enzyme activity (for proteins that have an enzyme function) Increased exposure of sulfhydryl groups (free cysteines) Loss of solubility

15 important questions on Proteins - Protein denaturation, protein stability

What happens with protein interactions upon heating?

Due to heating molecules start to vibrate, the energy outside of the system (T∆S) S = entropy
Then you have the stabilizing forces, the energy in the system(∆H).

∆G (unfolding) = ∆H-T∆S

When ∆H > T∆S there is no unfolding
When T∆S < ∆H you do get unfolding

What happens to a protein after heating?

It can go back to the native state
Or it goes back to an unnative state (denatured)

What happens with proteins during spray drying?

The denaturation temperature increases with decreasing water activity, consequently, unfolding does not necessarily take place during spray drying.

What is the effect of pH on proteins?

Gives reversible unfolding.
Proteins are more stalbe at ther IEP
extreem pH lead to unfolding

What happens to the protein at a very alkaline pH?

hydrolysis of peptide bonds
deamidation of Asn and Gln
S-S reshuffling
destruction of SH

Because of this there is more unfolding at high ph then at low ph

Why is a protein most stable at the IEP?

There is the same amount of + as - charge (THERE STILL IS CHARGE!) These can form a interaction and stabilize a protein.

If you go far away form the IEP you will have mostly negative or positive charge that repels each other and denatures your protein.

What is the effect of unfolding of detergents?

The detergents bind to an unfolded protein and changes there equilibrium. This makes that the protein prefers to stay in the denatured state.

This already happens at 3-8 mN.

What is the effect of organic solvents on protein unfolding?

They weaken the hydrophobic interactions
Enhance the formation of peptid H-bonds (therefore increases the strength of the secondary structure)
Enhance electrostatic interactions: attraction/repulsion.

However if the concentration becomes to high denaturation occurs.

What is the effect or organic solutes (sugars) on protein unfolding?

Solutes formation of H-bonds whit water molecules
This results in 'destructuring' of water
This results in solubilisation of hydrophobic AA residues
This results in unfolding

What is the effect of an unfolded protein?

Can lead to:

loss of enzyme activity (for proteins that have an enzyme function)
Increased exposure of sulfhydryl groups (free cysteines)
Loss of solubility

How does intrinsic fluorescence work?

You have AA that have a fluorescent spectrum.
Tryptophan is one of them. This is mainly present in the hydrophobic core of the protein. Tryptophan gets less fluorescent when it is in a hydrophilic environment compared to a hydrophobic environment.
This makes that you have more fluorescent absorbance in a native protein compared to a unfolded protein.

How will the presence of phenolic compounds (for instance as impurities during protein extraction) affect the fluorescence spectra?

In intrinsic fluorescence we use the fluorescent properties of Tryptophan, Tyrosine and Penylalanine. Thyrosine is actually also a phenol. Consequently, it can be expected that the phenolic compounds (non-protein phenols) also have some absorbance at 280 nm. If that is the case, then most likely they will also provide a fluorescence intensity. At least, this means that the fluorescence spectra will be influenced and cannot be interpreted to be only due to proteins.

Why is the chance of a protien folding back into its exact native state close to 0, but the energy state of the native state lower then the unfolded state?

Not only the energy level (∆H) but also the entropy (∆S) is important. The unfolded state can have many different structures resulting in a higher entropy that overcomes the energy level (enthaply: ∆H) .

What methods to determine protein unfolding can be used to calculate the thermodynamic parameters?

DSC, IF, and CD.
This information can be used to calculate the:

Free energy (∆G unf)
van 't Hoff enthalpy (∆H unf)
Activation energy of unfolding (∆E a,unf)

What is determined by ∆G(unf) and what by ∆E(a, unf)?

∆G(a, unf) interments the final equilibrium between folded and unfolded states.

∆E(a, unf): determines how much energy is needed to start this process.

So in the picture indicates that a low concentration of urea makes that the proteins take more time to unfold at a certain temperature compared to high concentrations.

The question on the page originate from the summary of the following study material:

Advanced food chemistry

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Topics that are related to Proteins - Protein denaturation, protein stability

Proteins - Protein denaturation, protein stability

15 important questions on Proteins - Protein denaturation, protein stability