Biochem - protein folding and stability

What can protein folding defects cause?

• Many diseases are the result of defects in protein folding

• Cystic fibrosis involves misfolding and resulting lack of protein involved in Cl- transport across membranes

• Many neurodegenerative disorders involve abnormal protein aggregation

• Partly folded or misfolded polypeptides or polypeptide fragments may associate with similar chains, forming toxic aggregates

At what points are proteins stable?

• Proteins are held together by an accumulation of lots of weak forces

• Only marginally stable

• Proteins are denatured by extreme pH and high/low temperatures

• Not stable in organic solvents as this strips them of water

What type of process is protein folding?

• Equilibrium process

• Fold to their most energetically favourable conformation

• Interaction with solvent plays a major role in folding - will not fold in a vacuum

• Proteins fold spontaneously - native conformation is most energetically stable

What are the stabilising forces within proteins?

• Hydrophobic interactions

• Ionic interactions

• Van der Waals interactions

• Hydrogen bonds

• Disulphide bonds

How do hydrophobic interactions promote a stable protein?

• Hydrophobic R-groups like to get away from water

• Hydrophobic groups will combine to minimise surface area interactions with water - like to be on the inside of globular proteins

• Hydrophobic amino acids have non-polar R groups (alanine, isoleucine, phenylalanine, tryptophan)

• Folding minimises the contact with water for the hydrophobic residues

• Water molecules will H-bond to other molecules in the solvent

• Will form an entropically unfavourable clathrate structure around the non-polar molecule

• Non-polar droplets will combine to reduce contact with polar water molecules

How do ionic interactions promote a stable protein?

• Electrostatic interaction between oppositely charged amino acid residues

• Lysine (+ve) can form an ionic bond with glutamate (-ve)

• Removal of the solvation shell (water molecules around the charge) is needed to break the ionic bond

• Cost of breaking ionic bonds is superceded by creating H-bonds with water to hydrate the molecules

How do van der Waals interactions promote a stable protein?

• Non-covalent interaction between electrically neutral molecules due to electrostatic interactions between permanent or induced dipoles

• Much weaker than ionic bonds but there are many of them

• Interactions have a positive and negative component

• Attractive and repulsive forces determine the optimal van der Waals radius (often called van der Waals distance)

• Optimal packing maximises van der Waals interactions

How do hydrogen bonds promote a stable protein?

• Electrostatic interactions between a weakly acidic donor (D) group and an acceptor (A) atom that has a lone pair of electrons

• H atom is between two electronegative atoms

• Many H-bonds in a-helices and B-sheets

• Any molecule that forms H-bonds to each other can also form H-bonds to water molecules

• H-bonds between two molecules dissolved in water is relatively weak due to competition with water

How do disulphide bonds promote protein stability?

• Mainly found in exported proteins or proteins that need to be stable at high temperatures

• Covalent bond between two cysteine residues - form a cystine residue

• Only covalent bond found to hold the polypeptide in its correct form

• Found mainly in proteins exported from the cell and proteins that require high thermostability

• Formed under oxidising conditions, can be broken under reducing conditions

What was Anfinsen’s experiment on folding?

• Added 8M urea and B-mercaptoethanol to a ribonuclease to chemically denature it

• When B-mercaptoethanol and urea were removed slowly by dialysis the protein refolded and formed correct disulphide bridges

• Protein returned to 100% of prior activity

• When only B-mercaptoethanol was removed cysteines formed random disulphide bridges

• When urea was removed afterwards only 1% of its activity was restored

• Formed a scrambled ribonuclease with random disulphide bonds

• Trace amounts of B-mercaptoethanol allowed the protein to relax and reform the correct disulphide bonds and restore its activity

• Concluded proteins fold simultaneously and the sequence dictates structure

What is the Levinthal Paradox related to protein folding?

• Assume each amino acid has 10 possible conformations

• A polypeptide of n amino acids has 10^n possible conformations

• Assume a protein can explore a new conformation every 10^-13 seconds

• A protein with 100 amino acids would take 10^87 seconds to fold

• Proteins must fold to their native conformations by directed pathways rather than random search methods

How is protein folding achieved in the cell?

• Correct protein folding is helped by other proteins (Chaperonins)

• Prevent aggregation of unfolded proteins in the cell

• Provide an environment for the protein to fold in