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
Examples of globular and fibrous proteins
Hb, enzymes, insulin and Ig = globular
Collagen, elastin, kerati, fibrin = fibrous
What is a filamentous protein?
Quaternary structure
NOT the same as fibrous proteins
What is the general structure of a globular protein?
Quite compact
Water is largely exclused from their interiors
Empty space and cavities exist
Van der Waals volume to molecuular surface volume = 0.75
When are motifs described?
Structural biology
What is a sequence motif?
Particular primary sequence that is characteristic of a specific biochemical function
What is a functional/structural motif?
Set of contiguous secondary structure elements = either have a specific functional significance or define a portion of an independently folded domain
What is the hierarchy of protein motifs?
Simple motifs
Complex motifs
Domains
Give examples of simple and complex motifs
alpha = Helix-Loop-Helix, HTurnH, leucine zipper
beta = β-hairpin, β-meander
mixed = βαβ and zinc finger
Ig-fold, Rossmann fold
P-loop
TIM barrel
What is the HLH motif’s main function, and where is it found?
Calcium binding in signalling and muscle contraction proteins
Serves as molecular switch in response to rise in cytosolic Ca2+ levels
What is HLH structure?
Two alpha helices = like index and thumb with calcium in the middle
What conformation are HLH normally found in?
In pairs normally
What is the bHLH motif’s main function, and where is it found?
DNA binding
One of the largest families of dimerizing TFs
Has loop allowing flexibility and connecting the two helixes (small and large)
Larger helix, typically, contains basic amino acids that facilitate DNA binding
Penetration of this helix into MAJOR DNA groove allows tight binding and recognition fo specific DNA sequences
What is the difference between HLH and bHLH? ***
HLH doesn’t do DNA-binding
bHLH is a specialised version of HLH with basic region for DNA binding and transcriptional regulation
What is the HTH motif’s main function, and where is it found?
DNA binding domain in many proteins that regulate gene expression
Similar to bHLH motif but has SHORTER LINKER
Name two other DNA-binding motifs
Leucine zipper = dimerization of two specific alpha helix monomers
Homeodomain = 60aa long domain composed of 3 alpha helixes
Name 4 beta motifs
Beta-hairpin = anti parallel
Beta-meander
Greek Key
Beta-sandwich
What is the role of beta-sandwich?***
Efficient packing of non-polar residues inside core
Very stable = good for creating functional sites
What is the function of βαβ motif and where is it found?
Most common mixed motif
Allows formation of parallel beta-sheets, binds nucleotides in the Rossmann & P loop folds
Also allows efficient packing of non-polar residues inside the core
What does the 1st loop often participate in, in βαβ motif?
Ligand binding of protein binding sites
What is the structure of a zinc finger?
2 cysteines and 2 histidine residues in this sequence
Forms ligands to a ZINC ion, whose coordination is essential to stabilize the tertiary structure
What is the role of zinc finger motif?
DNA-binding motif
What is more conserved than what?
Structure is more conserved than sequence
What is a superfold and where does it appear?
Highly common complex fold in proteins
May appear in proteins that have little sequence similarity
Examples of superfolds and what motifs they are based on
Ig fold = based on β-sandwich motif
Based on βαβ motif
- Rossmann fold
-P-loop
-TIM barrel
Explain the Ig fold structure ***
What is the function of a TIM barrel?
Mainly in enzymes catalysing diverse reaction
May act as scaffold or in catalysis
What is the structure of the TIM barrel?
Barrel centre not hollow
Densely packed with non-polar residues = provides shielding from the external env
Salt bridge network in barrel center
What is the structural evolution of TIM barrel?
Possess 2-fold, 4-fold, or 8-fold internal symmetry
Suggest they evolved from ancestral motifs
What mechanism did TIM barrels evolve by?
VIA gene duplication and domain fusion
What is the importance of different domains?
Domains often have specific functions, so having different ones confers FUNCTIONAL COMPLEXITY to proteins
Class I fusion glycoproteins structure
Homotrimer of single-transmembrane proteins
Class I fusion glycoproteins DOMAINS
Receptor-binding domain = rich in alpha-helices and hydrphobic fusion peptides located near N-terminus
Membrane-fusion domain = undergoes conformational change usually controlled by pH
Transmembrane domain = hydrophobic regions so that they prefer to be inserted into the cell membrane
Give an example of Class I fusion proteins
HIV-1 glycoprotein (GP160)
Influenza virus hemagglutinin (HA)
SARS-CoV-2 Spike protein (S)
Ebola virus gycoprotein (GP)
