Protein folding and stability Flashcards
Hydrogen bonds, disulphide bonds, ribonuclease refolding, interactions.
Cystic fibrosis involves misfolding and resulting lack of a protein involved in chloride ion transport across membranes.
Many neurodivergence disorders involve abnormal protein…
Aggregation.
List a few of these neurodivergence disorders.
Prion diseases (creutzfeldt-Jakob disease)
Alzheimer’s disease
Parkinson’s disease
How are toxic aggregates formed?
Partly folded/misfolded polypeptides or fragments may sometimes associate with similar chains.
How are proteins held together?
By an accumulation of weak forces.
Why are proteins described as only marginally stable?
They are not that much more stable than their unfolded states.
How are proteins normally denatured?
By extremes of pH (most only stable from pH 5-9).
By high temperatures.
Proteins are not usually stable in organic solvents.
Why?
This strips essential water.
What is protein folding based on?
Primary structure.
Protein folding is difficult to accurately predict based on…
Amino acid sequence.
What kind of a process is protein folding?
equilibrium
How do proteins fold?
To their most energetically favourable conformation and spontaneously.
What external factor has a major role in folding proteins?
The solvent.
When is a protein’s contact with the solvent maximal?
In unfolded states.
List all the forces and interactions that stabilise the 3D structure of proteins.
- hydrophobic interactions
- ionic interactions
- van der waals interactions
- hydrogen bonds
- disulphide bonds
Why do proteins tend to be quite stable if they have disulphide bonds?
These are the only covalent interactions.
Why are hydrophobic amino acids found inside of globular proteins? Why do pro
They repel water.
Hydrophobic amino acids have side chains that are…
non-polar.
What kind of core do proteins usually have?
Hydrophobic
When do clathrate structures of water molecules form?
During the hydrophobic effect water molecules form a cage like structure when they hydrogen bond with each other but (oil) droplets limit hydrogen bonds.
What does protein folding do for hydrophobic residues?
Minimises the contact with water for the hydrophobic residues.
What is a salt bridge?
Bonds between oppositely charged residues that are sufficiently close to each other to experience electrostatic attraction.
For instance, an amino acid with a positive side chain can form an ionic bond with an amino acid with a negative side chain.
For an ionic bond to form, it requires the removal of a…
solvation shell (where water molecules meet the polypeptide).
Why?
To offset (balance) energy liberated (set free) when bond is made.
What are van der Waals interactions?
Non-covalent interactions between electrically neutral molecules arising from electrostatic interactions between permanent dipoles and/or instantaneous dipoles and/or induced dipoles.
Describe the formation of London dispersion forces (a type of van der Waal interaction).
Chance charge separation creates an instantaneous dipole and a second molecule has a charge separation induced by the first molecule.
Positive side of first molecule attracted to negative side of second molecule.
How do van der Waals forces compare with ionic interactions within a protein?
Much weaker (but there are many of them within a protein).
Interactions have a positive component and a…
negative component.
What happens when molecules get too close?
Electron clouds repel.
What is the van der Waals distance?
(If confused, look at PowerPoint).
The optimal distance between 2 atoms.
What is the net force a result of?
The Leonard-Jones potential (energy = A/r^12 - B/r^6).
r^-12 = repulsive energy
r^-6 = attractive energy
How do you find the optimal distance on a graph?
Distance (in angstroms) from 0 to when net energy (in kcal/mol) line reaches lowest energy point on graph.
What determines the optimal van der Waals radius?
Attractive and repulsive forces.
Order the following van der Waals forces in terms of strength.
interactions between permanent dipoles > dipole-induced dipole interactions > London dispersion forces
What can maximise van der Waal interactions between atoms?
optimal packing
What are hydrogen bonds?
Electrostatic interactions between a weakly acidic donor, D, group (hydrogen partially letting go of) and an acceptor atom, A, that bears a lone pair of electrons.
In a hydrogen bond, the hydrogen atom is sandwiched between…
two electronegative atoms (usually O or N).
There are many hydrogen bonds in alpha-helices and beta-sheets.
What is the ideal bond length?
2.7 - 3.1 Å
How many residues is an alpha helix turn?
3.6
What is the pitch distance of an alpha helix (the distance the helix rises along its axis per turn)?
5.4 Å
How is hydrogen bonding arranged in a helix to result in the optimal length 2.8 Å?
The peptide C=O group of the nth residue points along the helix towards the peptide N-H group of the (n+4)th residue.
Where do the R-groups point in an alpha helix?
outwards
Any molecules that can form hydrogen bonds with each other can from hydrogen bonds with…
water molecules.
Due to competition with water molecules, the hydrogen bonds formed between two molecules dissolved in water are relatively…
weak.
What are disulphide bonds?
Covalent bonds between two cysteine amino acids in the protein chain.
Only covalent bond found to hold protein in its correct fold.
What kind of proteins are disulphide bonds mainly found in?
Proteins which are exported from the cell and have to be stable at high temperatures.
Although some forces may be strong, why do they only make weak contributions to protein stability in the unfolded state?
Similar strength interactions are made with the solvent.
When are hydrophobic forces exposed to the solvent and shielded from the solvent?
Exposed = unfolded.
Shielded = folded.
When are charged groups solvated (interact with solvent) in the protein?
In the proteins unfolded state.
What happens to charged groups in the folded state of a protein?
Ionic bonds form within the protein.
What do van der Waals forces form between in terms of proteins in their folded and unfolded states?
Unfolded = protein and water.
Folded = within protein.
What do hydrogen bonds form between in terms of proteins in their folded and unfolded states?
Unfolded = protein and water.
Folded = within protein.
When are disulphide bonds present in terms of the state of a protein?
Only present in protein’s folded state.
Who conducted the ribonuclease refolding experiment?
Christian B. Anfinsen
What was the ribonuclease refolding experiment?
When beta-mercaptoethanol and urea are added, the ribonuclease is denatured so there is no enzymatic activity.
When beta-mercaptoethanol and urea were slowly removed, the protein refolded and correct disulphide bonds reformed giving 100% activity.
When only beta-mercaptoethanol was removed, cysteines formed random disulphides and upon subsequent removal of urea approximately 1% of activity is restored.
How many cysteines are in ribonuclease?
8
Using the question above, where does the 1% come from?
(If confused, look at summary sheet).
1/7 x 1/5 x 1/3 x 1/1 = 1/105 ~ 1%
Correct protein folding is helped in the cell by proteins called…
chaperonins.
Why do chaperonins exist if proteins can fold spontaneously?
To stop proteins aggregating with other unfolded proteins (not to tell proteins how to fold).
What is the Levinthal paradox?
Finding the native folded state of a protein by a random search among all possible configurations.
This would take longer than the apparent age of the universe.
How long does it take for a protein to fold?
Less than or equal to seconds.
How do proteins fold to native conformations?
By directed pathways rather than random search methods.
The protein forms secondary structure elements to make a core.
