Dr. M.Vera Ugalde (20%) Flashcards
Why protein folding is important?
Activity of the protein depends on 3D shape
Where does information for 3D-structure come from?
- Classic experiment: Christian Anfinsen.
1st part of the experiment:
-> Ribonuclease A (124 residues with 4 disulfide bonds)
- Purified ribonuclease, can measure activity.
- Add Urea to denature (unfold) the protein, and βme (reducing agent) to break disulfide bonds.
- Remove urea then oxidize: full activity - protein folds by itself before correct disulfides form.
- oxidize then remove urea: no activity: random disulfides form and prevent folding to the native state.
2 part of the experiment:
- Add βme and activity slowly recovers -> breaking of random disulfides allows folding to proceed.
- the folding process is spontaneous, native structure is determined by primary sequence.
Afinsen’s Dogma
1) Conclusion #1 ->
- It states that the native structure of a protein is determined solely by its amino acid sequence, therefore by genetics.
- conformation of protein is unique
2) Conclusion #2 ->
- folding is spontaneous in principle
- native conformation has the lowest free energy
3) Conclusion #3 ->
- stability depends on environment
How does the protein folding process proceed?
- Cyrus Levinthal
- Rotation of backbone at Cα
- Assumption: 3 possible angles for phi and psi
What is Levinthal’s Paradox?
- The concept is that it highlights the improbability of proteins folding by randomly sampling all possible configurations.
- Assumption: 3 possible angles for phi and psi
- Dipeptide: 3*3 = 3^2
- Tripeptide= 333*3 = 3^4
- Peptide with 100 residues = 3^200 - 10^100
- single bonds reorient at a rate of 10^13/sec (overestimate)
- complete sampling will take 10^100/10^13s^-1= 10^87 sec
- present age of the universe is around 20 billion years = 10^18 sec
- but proteins can often fold in less than a few seconds
- it cannot be a random search for all possible positions
What is the Golf Course Energy Landscape?
- Horizontal coordinates: conformation of the polypeptide
- Vertical coordinate: internal free energy of the conformation
- If polypeptide randomly searches through all equally possible conformations, landscape will be flat.
THIS is wrong because different conformations have different energies.
Conformations closer to native state have lower free energies.
Free Energy of Conformations
- folding is a complex process (different free energy conformations)
- completely unfolded conformations have no internal contacts, and high free energy
- intermediate conformations with internal contacts have lower free energy
- free energy decreased as more internal contact’s form
What is the ‘Funnel Energy Landscape’?
- Folding proceeds through intermediates with increasing stability
- Free energy decreases (height above N)
- conformational freedom decreases (width of funnel)
- top of funnel: there are many different unfolded conformations with few internal contacts and high free energy.
- lower in funnel: folding intermediates have conformations that resemble and converge on the native state.
Folding Pathways
- Folding is speeded and guided by the rapid formation of local interactions.
- First set of interactions determine the further folding of the peptide.
- For any starting point on the funnel, there are ordered, kinetically accessible pathways to the native state. Explains how folding is possible on biological time scales.
- We consider the dynamics of folding in terms of changes in the internal free energy.
What is Gibbs Free Energy equation?
The maximum amount of energy present in a thermodynamic system that can be used to perform work at a constant temperature and pressure.
Enthalpy
- The measure of how much energy is released or absorbed during a chemical reaction.
- Enthalpic contributions are the formation of bonds (Hydrogen and ionic bonds, dipolar interactions, and cysteines disulfide bond)
Entropy
- State of disorder or randomness
- Entropy contribution:
1) Disorder of polypeptide decreases as folding proceed (not entropically favourable)
2) But hydrophobic interactions are entropically favoured by the movement of water molecules.
Hydrophobic Effect is Entropic. Explain?
- Entropic, it is driven by the water disorder, this is energetically favourable.
- Water molecules H-bond randomly with each other and polar residues - high entropy.
- They cannot H-bond with exposed hydrophobic residues, and instead form a rigid “cage” with each other - low entropy.
- When hydrophobic residues are clustered together, water is released from cages - high entropy.
What is the effect on Gibbs Free Energy during folding reactions?
△G<0 △H< T△S
- during folding, increased bond formation is balanced by decreased entropy of polypeptide.
- hydrophobic effect: increased entropy of water is enough to keep proteins stable.
- most normal proteins are marginally stable (sensitive to stress and mutations)
- △G of a domain around -50kJ/mol
- ATP hydrolysis: -30 kJ/mol
In essence, during protein folding, the decrease in free energy (ΔG < 0) comes from the balance of bond formation (negative ΔH) and the increase in water entropy (positive ΔS), even though the protein’s own entropy decreases.
Gibbs Free Energy (ΔG):
ΔG < 0: The reaction (like protein folding) is spontaneous and favorable.
A negative ΔG indicates that the products (folded protein) are more stable than the reactants (unfolded protein).
Enthalpy (ΔH):
During folding, bonds (like hydrogen bonds, van der Waals forces, etc.) form, releasing energy and making ΔH negative (exothermic).
Entropy (ΔS):
Folding reduces the entropy of the protein itself (less disorder as it goes from unfolded to folded).
However, the hydrophobic effect plays a crucial role. Nonpolar regions of the protein tend to fold inward, pushing water molecules outward, increasing the entropy of the surrounding water. This increased entropy can help compensate for the decreased entropy of the folded protein.
Hydrophobic Effect:
This is the tendency of nonpolar substances to minimize their contact with water. When proteins fold, hydrophobic residues are buried, leading to a more stable structure and increased entropy of the surrounding water, which favors folding.
What are the Structural Models of Protein Folding?
1) Framework Model (sequential):
- Secondary structures form first, then assemble into tertiary structure.
- Enthalpy driven, but ignores hydrophobicity.
2) Hydrophobic Collapse:
- hydrophobic core is buried first, then structures form around it.
3) Nucleation-Condensation:
- folding begins at one site on polypeptide with hydrophobic and enthalpic interactions.
- secondary and tertiary structures build outwards around the starting site.
- combination of sequential and collapse models, may be most common.
What is the typical folding process?
1) Begins with a large set of unfolded conformation with similar energies.
2) Proceeds quickly to compact molten globule intermediates (Collapse & Nucleation)
3) Slower stepwise formation of structure (condensation)
4) Discrete folding intermediates
5) Native Stricture
Like protein structure, folding is hierarchical - local secondary structures, then tertiary structures.
What is the Molten Globule?
- They are partially folded states
- Compact, partly organized, but flexible folding intermediate
- Not a single structure, but an ensemble of rapidly interconverting structures
- Most hydrophobicity is covered up, but interior is liquid-like and unstable
- Has many of the secondary structure elements of native state, but few tertiary structures
- loops and surface side chains may be disordered
- can sometimes capture molten globule by removing cofactors or metal ions needed for stability, or with mild denaturants
What are the folding time scales? from unfolded to molten globule? from final intermediate stages to native state?
1) Unfolded —> Molten globule (Fast 5 -1000 ms)
- secondary structure becomes stabilized, tertiary contacts begin to form
- side chains begin settling onto their native conformations
- protein is rapidly interconverting between an ensemble of closely related structures
2) Final intermediate stages —> Native state (slow, can take several seconds)
- complex motions required to attain relatively rigid core packing, hydrogen bonding, while expelling remaining water molecules from core.
Compare and Contrast Unfolded protein, Molten Globule and Native state.
Kinetically Trapped Intermediates
- Some folding pathways have energetic barriers
- Some intermediates (I1) can have low free energies, close to the native state (N)
- Transition state (T) has unfavoured conformation with high free energy
- I1 requires sufficient kinetic energy in polypeptide to proceed past T
- Polypeptides can remain in such kinetic traps for a long time
Kinetic Traps
- Structural interpretation: intermediates that have formed incorrect structures
- Transition state: requires partial unfolding to re-start folding with correct structures
- An intermediate with very low free energy (I2) and a high barrier may never progress to N.
- the functional form of the protein may then be the intermediate - the conformation with the lowest accessible free energy.
What chemicals denature proteins?
- Urea, guanidine, thiocyanate: cause unfolding at high concentrations (6 M)
—> Chaotropes.
They are not detergents like SDS (hydrophobic and charged ends) - Chemist discovered that different ions could increase or decrease the stability of proteins -> Hofmeister series of ions.
- Non-ionic solutes, like urea, later found to have similar properties.
Folding/Aggregation Landscape
- aggregates are stabilized by intra- and intermolecular hydrophobic interactions
- irreversible on short time scales and thermodynamically stable
- stability increases with aggregate size, and becomes more stable than native state
- most aggregates are amorphous: random conformations
- amyloid fibrils: proteins or fragments take on alternate repeating structures, neurodegeneration.
Protein Folding Catalysts
- PDIs and PPIs lower the energy barrier
of transition states that limit the rates of
folding - Comparable to enzymes
- Unlike chaperones, they recognize
specific features of polypeptides
