L4 Flashcards
How can proteins adopt their complex structure by themselves?
It should take longer than the ago of the universe but only takes a few milliseconds – why?
- Folding of proteins, protein-folding problem
- Levinthal’s paradox, Anfinsen’s experiments
- Conformational entropy, hydrophobic & polar forces
- The balance of forces in protein folding
- Energy landscaped & folding proteins on the golf course
What is the protein folding problem?
Most natural sequences will fold into a unique stable structure
- By what pathway does the protein adopt its native conformation?
- What is the physical basis of their stability?
- Why does the aa sequence determine 1 3-dimentional structure & not another?
- Given the aa sequence of the protein, can we hope to predict its 3-dimentional structure?
What was Anfinsen’s research group?
The Armour hot dog company purified 1kg of bovine ribonuclease A & offered 10mg lots free to scientists such as Christian Anfinsen
Studied the unfolding of Bovine Pancreatic Ribonuclease
He wanted to discover which pairs of the 7 Cys residues formed SS bonds
Tried to break bonds selectively (high concentrations of urea) to unfold the protein & reducing agents (mercaptoethanol, thiosulfate, dithiothreitol – DTT)
What did Anfinsen’s research show?
In 1 experiment, denatured solution was left exposed to air & its activity was regained afterwards – the protein could refold by itself
Atmospheric oxygen has oxidised the SH groups to SS & correct pairs had formed spontaneously
Protein folding information resides in the amino acid sequence
What was the Anfinsen experiment?
- Disulphide bridges were broken using urea
- Then remove the urea first to allow protein to reform its secondary structure
- Then oxidided it to reform the disulphide bridges
The control experiment starts of the same and the only difference is when then oxidise it fist & then remove the urea
- Forms all the wrong disulphide bridges
- Leads to scrambled proteins
- Removing urea causes the native state
What is the Levinthal paradox?
Because of the very large number of degrees of freedom in an unfolded polypeptide chain, the molecule has an astronomical number of possible conformations
Random search is not the way proteins fold
Proteins fold by following pathways involving partially folded intermediates
What are the time scales of protein folding?
Secondary structures: us – ms
Tertiary structures: ms – s
So there are 2 stages of protein folding: EARLY & LATE
What are the 2 stages of protein folding?
Early
Late
General conformation of a folded protein
- Low free energy
- Non-polar residues are buried
- Charged residues are exposed
- Exposure of non-polar atoms are balanced by polar atoms
- Low water content - hydrophobic interior
- Fixed positions of core atoms BUT conformational change possible – mobility of loop regions & exposed side chains
- Able to fold cooperatively
- Proteins denature with temperature
What forces stabilise the folded protein?
Hydrophobic effect
Ionic interactions
H bonds
Van der Waals
Hydrophobic effect in UNFOLDED proteins
Expose non-polar surfaces to water
This means water molecules have reduced H bonding opportunities & have restricted freedom of movement
This means that the water molecules are more highly ordered
Hydrophobic effect in FOLDED proteins
Expose a polar H bonding surface which also allows water to retain its mobility
Water is more disordered
The more disordered, the higher the entropy, S
Balance of energy is given by ∆G = ∆H – T∆S (Gibbs free energy)
Entropy increases when proteins fold & tis is due to water being released
Gibbs free energy in folded proteins
∆G = ∆H – T∆S
- ∆H is enthalpy – bonds involved in the system
- T is the temperature in K
- ∆S is the change in entropy
- To fold it has to go from a high energy state to a low energy state = a negative ∆G
- As the protein folds it squeezes any water molecules out from within it
- & since ∆H = 0 for protein folding then ∆S must be positive
Ionic interactions in FOLDED proteins
Can get interaction between positive & negative side chains & this is governed by Coulombs law
What is Coulombs law?
An experimental law of physics that quantifies the amount of force between two stationary, electrically charged particles
F = q1 x q2 / r^2 x D
r = distance
D = dielectric constant
q1 & q2 = 2 charges involved
It shows that the charge interaction is inversely proportional to r2 so only gets strong when the distance between each charge is short
