Protein folding

Protein folding

1. Central dogma

   • DNA becomes RNA forming a sequence which folds to enable a protein to fulfill its function
   • Native structure of the protein fulfills its function
     • Must be in a cellular environment
   • A protein chain folds in a specific way
     • Secondary and tertiary structures
     • Sequence defines fold
       • Sequence determines 3D structure
       • From an amino acid sequence you could guess the fold
       • A small protein of 101 amino acids has 9^100 possible conformations
       • Protein fold defines function
         • Free energy of unfolding
           • DELTAG(D-N) = GD - GN
           • To measure the differences the process needs to be in equilibrium
           • The equilibrium needs to be disturbed and determine the fraction folded
             • Denaturant urea is best
             • Temperature can be used but the process may not be reversible
               • Can cause protein misfolding
       • A ribonuclease solution will break all disulphide bonds and denature the protein giving its polypeptide chain structure
         • Another solution can reverse this and it will go back to its folded structure
       • Environment is an important folding factor

2. Proteins are marginally stable

   • Stability of denatured and native states is due to sum of non-covalent interactions
   • Folding is thermodynamically controlled
   • Native state is thermodynamically stable
   • Folding must occur rapidly

3. Levinthal's paradox

   • Sequence tells you what structure is likely to be
   • Folding cannot occur by the protein trying every different conformation
     • Take too long!
   • Folding must occur by intermediates

4. Pathway is needed!

   • Proteins must fold via pathways
   • Cut down the number of conformations the protein needs to search in native state
   • Folding funnels
     • Consider the energy landscape of folding
     • If protein folding was random then the diagram would be flat with the same energy
       • Protein would eventually find native state and fall down the middle
         • Takes too long

5. Hierarchical protein folding mechanism

   • Simplifies the search for native state
   • Uncouples formation of secondary and tertiary structure
   • Framework model
     • Secondary structure is separated followed by the tertiary structure
       • Requires intermediates
         • Reduces size of conformational space a protein has to search through
           • Denatured state to begin
             • Intermediate
               • High energy state of molten globules
                 • Much less stable and eventually ends up at low energy native state
   • Classical nucleation model
     • An element of secondary structure that nucleates the rest of the structure which allows formation of tertiary structure
   • Hydrophobic collapse model
     • All hydrophobic amino acids in the centre of the protein drive folding
       • Requires intermediates
   • All models rely on local interactions and sequence

6. Alternative view: no intermediates

   • Energy level is so high that you can't see the intermediate
   • CI2 (random protein)
     • Not necessary to have a stable intermediate
     • Protein goes straight from denatured to native state
   • Intermediates might slow folding
   • Intermediates may result in misfolding or an off pathway
   • No matter what state the protein is in, it will always fall back to the same native state as it is most energetically favorable
     • Folding funnels consist of lots of random highs and lows
       • Suggests possibility of multiple folding pathways
       • Bumps show that proteins could get stuck which could represent protein misfolding

7. Lysosyme

   • Small protein
   • Contains some disulphide bonds
   • Begins denatured and forms a collapsed state
   • There are a number of ways to get to the native state
     • Can be slow or fast pathway

8. Measuring protein folding and stability

   • Need to consider
     • How to monitor population of denatured and native
     • How to change populations of denatured and native
     • How to analyse results
   • Stability of protein is expressed as free energy of unfolding
   • Protein folding probes
     • In vitro assays in cuvette
     • In vivo assays under a microscope
     • Fluorescence will tell you about chromophores in protein
       • Can find buried or exposed amino acids in the protein
       • Unfolded state
         • Trp is exposed giving a particular fluorescence signal
       • Folded state
         • Trp is buried and gives different fluorescence signal
     • Emission spectra
       • Fix the excitation wavelength and scan the monochromator across fluorescence wavelengths
     • Unfolding proteins by denaturants
       • Urea most commonly used
       • GuHCL a much strong denaturing condition
       • Solvents increase the solubility of polar and non-polar side chains and stabilse the denatured state
       • Other denaturants
         • Temperature
         • pH
         • Salt
         • Pressure
   • Differences in protein folding between mutants
     • We know if a particular part of the molecule is important for folding
       • Mutate this part of protein and measure folding again
     • If free energy is different then we can tell the amino acid was important for protein folding

9. Experimental timeline and problems

   • To map folding reaction all states must be characterised
     • Mechanisms must also be characterised
   • Characterisation is often difficult
     • Dynamic, heterogenous, transient nature of non-native states
   • All possible states between denatured and native state must be investigated
   • Stable states are measured by equilibrium methods and protein engineering
   • Native state can use crystallography and NMR
   • Denatured state can be determined using NMR
   • Semi stable states must be trapped
     • Hydrogen/deuterium quench flow
       • Traps something as it's changing from denatured to native state
     • Sometimes conditions may be found where partly folded states can be observed by equilibrium methods
       • May not be relevant to pathway
     • Transient events can be followed by kinetic techniques
       • Stopped flow to follow protein folding
         • 2 solutions A and B can be mixed together and monitored during a reaction
         • A solution fills stopping syringe, plunger hits a block causing flow to be stopped instantaneously
         • Using appropriate techniques, the kinetics of the reaction can be measured in the cell

10. Representing protein folding

    • Chevron plot
      • Shows protein folding kinetic data in varying denaturant concentrations
      • Shows protein folding and unfolding
      • Cm is the denaturation midpoint
        • Either side are the limbs
          • Linear limbs
            • Straight lines
            • Suggests a 2 state model
          • Non-linear limbs
            • Curving
            • Indicates a non-2 state model of folding/unfolding
            • Suggests an off pathways intermediate
      • Chevron/V-shaped kinetics curve
        • Proteins are denatured as all parts of the protein are more soluble
        • Free energy of transfer to denatured state is nearly linear with concentration of denatured protein
        • TS in an intermediate structure between denatured and native states
          • TS stabilised by denatured state with respect to native structure
            • TS destabilised with respect to denatured state
          • Structure of proteins TS for folding can be analysed by combining kinetic and equilbrium data
            • Produces a ratio called PHI value
              • Value obtained by normalising
                • Free energy from folding kinetics
                • Free energy from refolding kinetics
                • Free energy from equilibrium data
        • Denaturant lowers activation energy of denaturation and raises activation energy of folding
          • 2 state model means intermediate is passed by quickly