Protein Folding

Protein Folding

Significance
1. Part of the central dogma
  1. DNA --> RNA --> Sequence --> Fold --> Function
    1. Interpretation: Sequence defines fold
      1. Example - a small protein of 101 amino acids
        In theory: 9^100 possible conformations
        BUT in practice there are not this many. Each amino acid contributes specific chemical properties to the protein
        This defines the way regions of the protein interact with one another and therefore the final fold
      2. Because we know a lot of protein sequences and the corresponding native fold, it is possible to predict a protein's fold based on its sequence
2. The native fold is the structure of a protein that allows it to fulfil its function
  1. i.e. it is not functional without this
3. Anfinsen, 1961
  1. The amino acid sequence of a polypeptide chain contains all the information required to fold the chain into its native, 3D structure
  2. Ribonuclease denatured with 8M urea and beta-mercaptoethanol
    1. Always folds back into its original conformation when allowed to reoxidise in air
  3. Small proteins
    1. Those which are larger are often more complicated and may require chaperones etc
Kinetics and stability
1. Stability of denatured and native states
  1. Due to the sum of all non-covalent interactions
    1. Between atoms of the protein
    2. Between protein and solvent
    3. delta-G[D-N] is the difference in the sum of two very large nunbers
      1. Free energy of unfolding
        In considering factors that contribute to the stability of proteins we have to consider both the denatured state D and the native state N.
      2. deltaGD - deltaGN
  2. Can determine the change in free energy on mutation
    1. m values
    2. Modify key parts of the protein and see what happens to the free energy
2. Folding is thermodynamically controlled
  1. Native state is thermodynamically stable
    1. The process is reversible
      1. Need an equilibrium which can be disrupted
        Can only determine stability in equilibrium conditions
        Many proteins can't be denatured reversibly!
        Proteins fold and unfold co-operatively
        The only two states at equilibrium are D and N
        How are these populations measured and monitored?
        Add different things to try and change the equilibrium conditions
        HOW do we change the populations of D and N?
  2. Entropy/enthalpy
3. Folding must occur rapidly
  1. Levinthal's paradox (1968)
    1. Folding not simply by protein trying every conformation until it finds the most stable one!
      1. Would take too long
      2. Unlikely to find the same conformation every time
    2. Small protein of 101 residues
      1. Each peptide bond, 9 main conformations
        Amount to the rotation positions around each of the two bonds adjacent to the peptide bond itself
        100 peptide bonds
        9^100 possible conformations
        Around 1090
        Ignores conformations due to side chains
  2. Time taken to switch conformation around 10^-13 sec
    1. If protein started folding at the time of the earth's origin it would have explored around 1.4x10^30 conformations
      1. Only a tiny proportion of those possible!
        => Folding must occur via intermediates in one or several defined pathways
        Cuts down the number of conformations that proteins need to search to find the native state
        Folding funnels
4. There can be more than one energetically stable state for a protein
  1. Amyloid
    1. Misfolded protein which is very stable
      1. Aggregation diseases
Hierarchical folding mechanisms
1. Uncouple the formation of secondary and tertiary structure
  1. Simplify the search for the native state
2. Framework model
  1. Sequential folding one residue at a time
3. Classical nucleation model
  1. Central piece forms and the rest of the protein folds around it
4. Hydrophobic collapse model
  1. All hydrophobic residues cluster in the centre of the protein
    1. Protection from the external aqueous environment
  2. The rest of the protein folds in around the hydrophobic residues
5. These models require intermediates!
  1. Reduces the conformational space a protein has to search through
    1. Solves the Levinthal paradox!
6. Alternative view: no intermediates
  1. Jackson and Fersht, 1991
    1. Chymotrypsin inhibitor 2
      1. Stable intermediates not essential for fast and efficient folding of a protein
        Extremely short-lived
  2. New model: condensation/nucleation
    1. Something happens in the centre of the molecule which causes the rest of the residues to fold in
Biased
1. Levinthal assumed unbiased search
2. Chemical properties of amino acids result in inherent preferences
  1. Folding is more like a funnel than a flat filter
    1. The native state is that which is most energetically favourable due to various intramolecular interactions
3. "Energy landscapes"
  1. Certain pathways require more energy than others
    1. A bit like a mountain range!
    2. Accounts for the possibility of misfolding
    3. Without the energy required to get out of "dips" could get stuck in incorrect pathways
      1. This would lead to misfolding
      2. Kinetic traps
Probes
1. Fluorescent amino acids
  1. Use spectrophotometry to track the fluorophore
    1. i.e. is it buried inside the protein or exposed?
      1. This is a good indicator of whether or not the protein has folded
  2. Try to observe fluorescence in a different direction to transmission
2. In vitro assays
  1. Cuvette
3. In vivo assays
  1. Under the microscope
4. Trp shifts when its environment changes
  1. Easier to understand folding in proteins rich in these residues
5. Tyr changes intensity
Unfolding by denaturants
1. How do they work?
  1. Preferential binding model
  2. Preferential solvation model
  3. Increase solubility
    1. Polar side chains
    2. Non-polar side chains
    3. Protein backbone
    4. Stabilise denatured state more than native state
      1. Larger surface area
2. Common denaturants
  1. GuSCN
    1. GuHCl
      1. Urea
        Most common
3. Other denaturants
  1. Temperature
  2. Pressure
  3. pH
  4. Force
  5. Salt
Experimental problems
1. Need to characterise all states along the way
  1. Also conversion mechanism between them
  2. Difficult
    1. Dynamic
    2. Heterogeneous
    3. Transient
2. Measuring D-state
  1. NMR
  2. SAXS
3. Measuring N-state
  1. Crystallography
  2. NMR
4. Options
  1. Trap semi-stable states
    1. Hydrogen/deuterium exchange quenched flow
    2. Sometimes partly folded states can be observed by equilibrium methods
      1. May not be relevant to the pathway!
      2. "molten globule" NMR
      3. Native state hydrogen exchange
      4. Peptide studies
  2. Stopped flow
    1. Pneumatic drive activation - two small volumes of solutions driven from high performance syringes through high efficiency mixer
      1. Resultant mixture passes through measurement flow cell and into stopping syringe
        Just prior to stopping steady state flow is achieved
        Solution fills the stopping syringe and the plunger hits a block
        Flow is stopped instantaneously
        Kinetics of the reaction can be measured in the cell
Representing protein folding
1. Chevron plot
  1. Protein folding kinetic data in varying denaturant concentrations
    1. Can depict protein folding or unfolding
      1. Folding in one direction and unfolding in the other
  2. Cm is denaturation midpoint
    1. Either side are "limbs"
      1. Linear limbs
        2-state model
        m-values
        Does NOT mean there are no intermediates!!!
        The intermediate states are not significantly populated
        "kinetically invisible"
      2. Non-linear limbs
        Non-2-state model
        Off-pathway intermediate?
        Artifact?
        Because it goes through more stages
  3. V-shaped kinetic curve
    1. Proteins denatured: all parts of protein are more soluble in denaturant
      1. deltaG of transfer to denatured state is almost linear with [denaturant]
      2. TS is the intermediate structure between denatured and native states
        Stabilised by denaturant with respect to native structure
        Destabilised with respect to denatured state
      3. Denaturant lowers activation energy of denaturation
        Raises activation energy of folding
        Stabilisation energy linear with [denaturant]
2. Phi value analysis
  1. Combine kinetic and equilibrium data to produce a ratio
    1. Phi value
    2. Analysis of a protein's TS for folding
      1. Change intermediates by introducing specific mutations
        Choosing mutations
        Properties
        Non-disruptive
        Conservative
        Should not change structure
        Should not change the polar nature of the side chain
        Good examples
        Ile --> Val --> Ala
        Phe --> Leu
        Thr --> Ser
        Asp --> Asn
        Poor examples
        Leu --> Val
        Trp --> Anything
        Very specific folding
        Provides a fluorophore
        Risky
        Pro --> ?
        ? --> Gly
        Gly --> ?
        Must be significantly destabilising to get reliable phi value
        Should make a large number of mutants in order to get a realistic picture
    3. Obtained by normalising either (deltadelta)G[int-N] from unfolding kinetics
      1. OR (deltadelta)G[D-int] from refolding kinetics
      2. OR (deltadelta)G[D-N] from equilibrium data

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	Created by Jen Harris about 11 years ago