Protein Folding Flashcards
Tertiary and Quaternary
What is Anfinsen’s dogma for protein folding?
Thermodynamic hypothesis
For a small globular protein in its standard physiological environment, the native structure is determined only by the protein’s amino acid sequence.
What is the role of heat shock proteins in protein folding and how do they do this?
Upregulated in response to stress
Bind to hydrophobic regions of unfolded proteins
Stabilising them to prevent misfolding and aggregation
What experiment did Anfinsen perform?
Denaturation of ribonuclease A (RNase)
What does urea do to RNase A?
Disrupts the non-covalent interactions = H bonds and hydrophobic interactions
What does β-mercaptoethanol (BME) do to RNase A?
Cleaves the 4 disulphide bonds reversibly
What happens to RNase A when remove urea and BME by dialysis, slowly in the presence of oxygen?
Sulfhydryl groups of denatured enzyme become oxidized by air and enzyme spontaneously refolded into a catalytically active form
What happens to RNase A when only BME is removed?
Disulphide bonds reform in the denatured protein
Results in scrambled RNase, only has 1% of enzymatic activity of native protein
This is because wrong disulphides formed pairs in urea
What happens to RNase when BME is removed first and then urea?
Enzymatically inactive protein because disulphide bonds have formed at random
How is RNase converted back to its fully active, native conformation?
Then trace amounts of BME is added to scrambled RNase with oxygen
The added BME catalysed the rearrangement of disulphide pairing until native structure was regained in ~10h
Why do proteins fold?
Native globular proteins are only marginally stable under physiological conditions
For 100-residue protein, it is more stable in folded than unfolded form
What does the top of the protein folding funnel represent?
Top represents the unfolded states, w high degree of conformational entropy
What does the narrowing of the protein folding funnel represent?
As folding progresses, the narrowing of the funnel shows decrease in number of conformational species present
What do the small depressention of the protein folding funnel represent?
Semi-stable intermediates which may slow the folding process
What does the bottom of the protein folding funnel represent?
Folding intermediates that have been reduced to a single native conformation
What is the role of chaperone proteins in protein folding?
They bind themselves to hydrophobic surfaces = reducing the possibility of two proteins or polypeptides binding and forming aggregates
They use ATP to facilitate forming of protein’s native state
Do proteins fold to the most entropically favourable conformation?
No, unfolded state has highest energy, so native states are more stable but not the most stable
Some proteins naturally occur in amyloid form but it is rare
Different shape of globular vs fibrous proteins
Globular are typically spherical, while fibrous are usually long and narrow
Different amino acid sequence of globular v fibrous proteins
Globular normally have irregular and wide rang of R groups
Fibrous tend to have repetitive and limited rate of R groups
Different function of globular v fibrous proteins
Globular are functional and carry out a specific biological function
Fibrous are structural = help maintain cell shape by providing a scaffolding
Different solubility of globular vs fibrous proteins
Globular are generally soluble in water
Fibrous are generally insoluble in water
Different resilience of globular v fibrous proteins
Globular are more sensitive to temperature and pH
Fibrous are less sensitive
