Viles L2 - Protein Folding Pathway and Stability

Viles L2 - Protein Folding Pathway and Stability

1. WHY STUDY FOLDING + STABILITY

   • 1. Correct fold required for function + stability (GFP)
   • 2. Folding like to many things: protein trafficking, cell growth + differentiation, cell cycle and immune response etc
   • 3. Failure to fold results in disease
     • CF, some cancers, amyloid diseases
     • Aggregation of misfolded proteins that escape quality control mechanisms is characteristic feature of diseases
   • Can study thermodynamics (stability) and kinetics of folding

2. Proteins fold due to intrinsic properties of aa sequence and contributing influences from crowded cellular milleux

3. LEVINTHALS FOLDING PATHWAY HYPOTHESIS

   • Folding kinetics
   • Small protein with 100 residues, each can adopt 3 possible phi, psi angles = 3(power 100) poss structured
     • Ramachandran diagram shows possible angles
     • would take longer than the time the universe has existed to fold
       • Folding takes a pathway (folding funnel) - cannot be random
         • FOLDING FUNNEL
           • Depicts thermodynamics of protein folding
             • Not physical describes energy of folding
               • Folding energy surface UNIQUE to every protein
           • This pathway is not smooth - local minima (bumps) protein can get stuck in
             • Follows path towards lowest energy native conformation
               • Protein can follow different pathways but gets to same final native topology
             • Protein sequence evolves so that it doesnt get stuck and folding remains rapid
           • Large proteins fold locally into domains + subdomains within same polypeptide chain
             • CD4 = Immune cell surface protein that HIV binds has 4 domains
           • UPGRADED TO FOLDING CUBE
             • Again structure tends towards lowest energy conformation
               • Tendency to retain partially folded intermediates of lower energy drives folding
             • Height of cube (y axis) = free energy, X axis = no native interactions, z axis = no residues involved
               • Top of cube = least stable denatured, bottom = most stable with most residues forming native interactions
             • Saddle point = TS/intermediate conformation
               • Once structure reaches this is overcomes slight energy barrier then rapidly folds after (steep trajectory)
               • In TS = key phobic residues have interactions similar to native structure
                 • In TS = Individual AA substitutions show small no/residues (3/4 key phobics) form nucleus around which remaining structure condenses
                   • These residues nucleate folding (unfolded - molten globule - folded)
                     • Can measure molten globule state with NMR as has milli-second movements
                       • Measurable state before protein reaches native fold

4. FOLDING DRIVEN BY HYDROPHOBIC FORCES OF KEY RESIDUES

   • EVIDENCE
     • Adding soluble residues to N + C terminus doesn't affect rate
     • Mutations of hydrophilic residues in surface doesn't affect rate or fold
     • Only changing key residues will affect folding
   • Phobic side chains bury early on
     • Reduces number of possible conformations
       • As side chains bury their polar backbone cannot H-bond to water so H-bond to each other = 2 structure
   • In primary sequence phobic residues are scattered not clustered
     • >1/2 buried in native folded state
     • Protein samples huge range of confirmations
       • They are selectively buried in right topology during rapid formation of molten globule/TS
         • Because native like interactions more stable and persistent

5. SECONDARY STRUCTURE + FOLDING

   • Helical proteins fold more rapidly as secondary structure contacts are local
   • B-sheet AA contacts more distant in sequence - take longer to find native topology

6. 4 DESCRIPTIONS OF FOLDING PATHWAY MECHANISMS

   • 1. FRAMEWORK MODEL
     • Elements of secondary structure form first, these diffuse and collide so tertiary structure forms
       • 2 structure - tertiary structure
   • 2. HYDROPHOBIC COLLAPSE MODEL
     • Hydrophobic + key residues bury first to form restricted molten globule intermediate, protein rearranges from this to form secondary and tertiary structure
       • Hydrophobic collapse - rearrangment - secondary - tertiary
   • 3. NUCLEATION PROPOGATION MODEL
     • Local interactions form small amount of native 2 structure that acts as nucleus for outward propagation of native structure
       • Nucleation (local) - propagation - native
   • 4. NUCLEATION/CONDENSATION MODEL
     • Presence of metastable nucleus (part 2 structure, part key phobics) that triggers folding once number of stabilising long-range interactions have built up
       • Then native structure forms rapidly - nucleus not fully formed in TS
       • Mix 1 and 2

7. PROTEIN STABILITY

   • Folding thermodynamics measured at EQUILIBRIUM
   • Folding is a reaction with equilibrium and free energy
     • K = Product/reactant = unfolded/folded
       • Eq constant not rate
   • If protein is causing disease may want to know why its unstable
     • Mutations
     • Copper
     • pH
   • DENATURATION CURVE
     • Simple 2 state unfolding normally
       • K = 1 @ MIDPOINT
     • % unfolded vs denaturant
       • More denaturant more unfolding
     • Folding is highly cooperative process
       • Sharp not gradual transition
         • At midpoint of graph half unfolded, half not, none partially folded
           • Different for multi-domain proteins
         • All or nothing rapid process
     • Multi-state unfolding
       • For large multi-domain proteins
         • Unfolding intermediate prior to completely unfolded state
     • Measures stability by amount of energy/denaturant required to unfold
       • Ribonuclease requires 8M urea and B-merceptoethanol to break disulphides
       • Most proteins need 8M urea + 6M GuHCl
     • Remove denaturant should refold (100% activity)
       • All info to specify structure + activity in AA sequence
     • DENATURANTS break non-covalent bonds
       • Urea + GuHCl interfered with H-bonds + hydrophobic effect (thought to replace water)
       • B-merceptoethanol has thiol groups to break disulphides
       • Heat + Pressure (forces water into core)
       • pH changes protonation of AAs so breaks electrostatic interactions + removes salt bridges
     • Measuring curve
       • want to know stability @ equilibrium (wait few hours before measuring)
       • Unfolding must be reversible which urea + GuHCl are
         • Thermal not as aggregates after
           • Aggregation associated with its own free energy so confuses measurements
           • Doesn't rapidly refold = aggregated
       • Disulphides also make it more complex
     • FREE ENERGY OF FOLDING FROM CURVE
       • Got equilibrium constant from denaturant graph
         • We want stability = free energy of folding
           • K proportional to free energy
             • Can use equation ... Free energy = -RTln(K)
               • So times K by (-RTln)
               • Produces linear graph
                 • Can extrapolate to no urea = hence native topology stability

8. WHAT DESTABILISES PrpC ?

   • Cu(II) reduces thermal stability of PrpC
     • With copper unfolds 10 degrees lower (50 degrees)
       • Shown by molar eliipticity measured by CD
   • Oxidation of methionine in protein core
     • Only 2M not 5M required
   • Acidic pH
     • @ pH4 prion forms intermediate rich in B-sheet then completely unfolds
       • @ pH4 forms amyloid faster than pH7 = destabilised
   • All detected by CD

9. DETECTION METHODS FOR UNFOLDED AT EQUILIBRIUM

   • As protein unfoldes folds these characteristics change
     • CD
       • Difference in absorption of left + right circularly polarised light = molar ellipticity over wavelength
       • Can monitor process under various conditions, pH, ionic strength, temp, copper
     • FLOURESCENCE
       • Aromatic AAs (Phe, Trp, Tyr) = intrinsic flouresent probes
         • Fluorescence occurs at longer wavelength (lower energy) than absorbance
           • Stokes shift = difference between band maxima of absorbance + emission
         • Intrinsic fourescence - nothing added
         • Fluoresce different if buried or solvent exposed
           • Can probe unfolding
             • Thermal denaturation of albumin monitored by fluorescent probes
               • Increasing temp changes energy + maxima
     • UV ABSORPTION
     • NMR
       • Very powerful
       • Can monitor changes in folding state for each AA
         • Peaks vanish/shift when environment changes (e.g. unfolding in pH)
           • Peaks restored when pH returned
       • TS molten globules broaden spectra - ms movements
     • SIZE OF PROTEIN (HYDRODYNAMIC RADIUS)
       • Dynamic light scattering, viscosity, diffusion
         • Use electrophoresis gel where protein is travelling through gel with increasing urea
           • Mutant/less stable protein unfolded at lower urea concentrations (earlier in gel)

10. EXCPETIONS

    • intrisically unstructured proteins
    • Metamorphic proteins (ensemble of native structues in equilibrium)