chem for bioscience

• Protein structure and folding

  • What structures are formed?
  • Why are they stable?
  • Why do proteins fold? Thermodynamics of folding
  • How do proteins fold? Pathways & kinetics
  • Can mis-folding cause disease? (e.g. prions & CJD; Alzheimer's Disease)
• Non-covalent forces that stabilise the folded protein structure

  • Hydrogen bonds
  • Electrostatic interactions
  • Van der Waals (or London) forces
  • Hydrophobic effect
• Disulphide bonds between cysteine residues are COVALENT bonds, not non-covalent
• Non-covalent forces that determine protein structure

  • Van der Waals forces
  • Electrostatic interaction
  • Hydrogen bond
  • Hydrogen bond
  • Hydrophobic interaction
• Bond enthalpy (kcal mol-1)

  • -0.03
  • -5
  • -3
  • -4
  • -
• Free energy change (kcal mol-1)


  • -
  • 1
  • -
  • 2.4
  • -
• A dielectric constant of 4 is assumed in calculating electrostatic energies
• Hydrophobic effect


  Water molecules in contact with a hydrophobic side-chain cannot form any type of non-covalent bond with that side-chain, and so they form tighter bonds with each other (water-to-water H-bonds). This "ice-like" water structure that surrounds each hydrophobic amino-acid side-chain can be released, and the water molecules allowed greater freedom, if the hydrophobic side-chains all cluster together in the core of the protein. Greater freedom means greater entropy, and thus the hydrophobic effect is driven by the tendency for water molecules to gain greater entropy.
• Hydrogen bonds each contribute only about -1 kcal/mol to the free energy of folding, not -3 kcal/mol, because in the unfolded state the carbonyl and amide groups can each form (weak) H-bonds with water molecules in the unfolded state (each worth ~ -1 kcal/mol)
• Electrostatic interactions each do not contribute -5 kcal/mol to the free energy of folding, because the charges are fully solvated (neutralised) by the polar water molecules in the unfolded state. The entropic contribution of 1 kcal/mol remains, due to the water molecules that are no longer forming ordered solvation shells and can go free.
• Entropy of folding the polypeptide chain

  • Entropy can be related to the number of states, or degrees of freedom of a system
  • S = k ln W, where k is Boltzmann's constant and W is the number of states
  • For a mole of protein, Sfolding = Snative - Sunfolded = R ln (Wn/Wu), where Wn is the number of native conformations (1) and Wu is the number of unfolded conformations (10^100 if 100 residues with 10 conformations per residue)
  • Sfolding = -0.44 kcal mol-1 K-1 at 300 K, or -132 kcal mol-1
• Balance sheet for protein folding

  • Hydrophobic: 40 buried residues at -2.4 kcal mol-1 each = -96 kcal mol-1
  • Hydrogen bonds: 40 bonds at -1 kcal mol-1 each = -40 kcal mol-1
  • Electrostatic: 3 salt bridges at -1 kcal mol-1 each = -3 kcal mol-1
  • Van der Waals: 100 interactions at -0.03 kcal mol-1 each = -3 kcal mol-1
  • Total enthalpy = -142 kcal mol-1
  • Entropy of chain folding = +132 kcal mol-1
  • Net ΔG for folding = -10 kcal mol-1
• The hydrophobic effect also drives protein-protein interactions, with a buried surface area of 100 Å2 (1 nm2) contributing 2.4 kcal/mol, or 0.024 kcal/mol/Å2 of buried surface area
• The principal driving force for protein folding (or protein-protein interactions) in aqueous solution is the increased entropy of the water molecules that results from burying hydrophobic amino-acid side-chains
• Protein Folding - Experimental Observations

  • Spontaneous folding to a single native structure
  • Fast process
  • Co-operative process
  • Thermodynamic measurements suggest ΔG ~ -10 kcal mol-1
  • Defined pathway or trial and error?
• Amino-acid sequence


  • Folded structure
  • Trial-and-error or folding pathway(s)?
• If a protein of 100 amino-acid residues has 10 conformations per residue, the total number of conformations is 10^100, which would take 10^80 years to sample at a rate of 10^13 per second. In reality it takes only 1 second - this is Levinthal's Paradox.
• The amino-acid sequence encodes both the protein structure and the folding pathway
• Bovine pancreatic trypsin inhibitor (BPTI) folding scheme


  Complicated, no single pathway, may involve non-native conformations
• Folding pathway for lysozyme

  α-helices form first, β-sheets later
• Protein folding in vivo: the role of chaperone proteins


  • Most proteins require "chaperone" proteins for folding
  • GroEL/GroES E. Coli chaperone protein complex
  • Folding cavity
  • Closed state - hydrophobic surfaces exposed, protein unfolded
  • Open state - hydrophobic surfaces hidden, hydrophilic residues exposed, protein folds
• Haemoglobin and sickle-cell anaemia

  • Haemoglobin tetramers pack together via the hydrophobic interaction of valine 6 in the beta-chain, leading to fibre formation
  • Fibres distort and rupture the red blood cell, causing anaemia
• Protein mis-folding and disease

  Electron microscope images of protein fibrils formed by A) Alzheimer's disease beta-peptide, and B) mouse prion protein
• The prion hypothesis


  • PrPC normal "cellular" form
  • PrPSc abnormal "Scrapie" form
  • PrP protein can exist in two inter-convertible conformations with identical amino-acid sequence, PrPC and PrPSc
  • Unlike PrPC, PrPSc can polymerise to form insoluble fibrils
  • PrPC has more α-helix & no β-sheet, susceptible to proteolysis
  • PrPSc has less α-helix & more β-sheet, protease resistant
• Proteins polymerise via beta sheet formation, forming amyloid fibrils that cause cell and tissue destruction in diseases such as CJD and Alzheimer's disease
• Cryo-EM structures of tau filaments from Alzheimer's disease were published on 13th July 2017
• Non-covalent forces that stabilise the folded protein structure

  • Hydrogen bonds
  • Electrostatic interactions
  • Van der Waals (or London) forces
  • Hydrophobic effect
• Disulphide bonds between cysteine residues are COVALENT bonds, not non-covalent
• Hydrogen bond


  Bond enthalpy -3 kcal/mol, free energy change -4 kcal/mol
• Electrostatic interaction


  Bond enthalpy -5 kcal/mol, free energy change 1 kcal/mol
• Van der Waals forces

  Bond enthalpy -0.03 kcal/mol
• Hydrophobic interaction


  Free energy change -2.4 kcal/mol for buried side chain of Phe
• The hydrophobic effect is driven by the tendency for water molecules to gain greater freedom (entropy)
• Hydrogen bonds each contribute only about -1 kcal/mol to the free energy of folding, not -3 kcal/mol, because the carbonyl and amide groups can form weak H-bonds with water in the unfolded state
• Electrostatic interactions each do not contribute -5 kcal/mol to the free energy of folding, because the charges are fully solvated (neutralised) by water in the unfolded state. The entropic contribution is 1 kcal/mol.
• Entropy of folding the polypeptide chain

  • S = k ln W, where k is Boltzmann's constant and W is the number of states/conformations
  • For a 100 residue protein, S_folding = -132 kcal/mol at 300K
• Entropy of folding the polypeptide chain

  1. S_folding = S_native - S_unfolded

  2. S_native = R ln 1 (only 1 native conformation)

  3. S_unfolded = R ln 10^100 (100 residues, 10 conformations per residue)

  4. S_folding = -0.44 kcal/mol/K at 300K
• The free energy of folding of a protein is typically only -10 kcal/mol, so proteins are very easily destabilised
• Hydrophobic effect for buried surface area

  0.024 kcal/mol/Å^2 of buried surface area
• The principal driving force for protein folding (or protein-protein interactions) in aqueous solution is the increased entropy of the water molecules that results from burying hydrophobic amino-acid side-chains