Proteins/amino acids

• What is the predominant ionization state of an amino acid at different pHs?

  • At very low pH: carboxyl and NH3+ is protonated
  • At pH 7: Zwitterionic form (carboxyl deprotonated, both + and - charges on amino acid)
  • At high pH: carboxyl and NH3+ deprotonated
• Fischer projection

  • horizontal bonds project outwards
  • vertical bonds project backwards
• Typical pKa values of ionizable groups in proteins

  • terminal alpha-carboxyl group: 3.1 (will be deprotonated at physiological pH)
  • glutamine: 4.1 (DP)
  • Histidine: 6 (DP)
  • terminal alpha amino: 8 (P)
  • Cysteine: 8 (P)
  • Tyrosine: 11 (P)
  • lysine: 11 (P)
  • Arginine: 12.5 (P)
• Peptide bonds link amino acid residues by their carboxylate and amino groups.
• Why do peptide bonds form if individual amino acids are lower energy?

  peptide bond may be thermodynamically unstable, it is kinetically stable
• Residues are numbered from which terminus of a polypeptide?

  amino
• Average weight for an amino acid residue?

  110 g/mol (Da)
• Primary structure

  linear polymer of amino acids residues with peptide bonds and is the sequence of the protein from the amino terminus to the carboxy terminus
• Is trans or cis conformation of peptide bonds more favourable?

  trans because of less steric strain
• Angles of rotation

  Phi ϕ: rotations of N-C alpha bond

  Psi Ψ: rotating alpha carbon-carbonyl bond

  Omega ω: rotation about the peptide bond - trans (180 °) and cis (0 °)
• Ramachandran diagram

  • The typical phi, psi for a residue in a beta-sheet is -80 deg, 100 deg
  • The typical phi, psi for a residue in a alpha helix is -60 deg, -60 deg
• Which amino acids form disulfide bonds?

  Cysteines are oxidized to form a disulfide bond with each other
• Alpha helices in proteins are typically left or right handed?

  Right handed
• how far away can atoms hydrogen bond within an alpha helix?

  4 amino acids away because chain is twisted
• Which way are the carbonyls pointing in alpha helices with optimum angles? What about beta sheet?

  Beta sheets: parallel

  Alpha helices: inwards and outwards
• Why is a phi angle of 90 and psi of -90 unfavourable?

  Steric clashes between carbonyl oxygen and R group, and amino group with R group
• In the main chain of an alpha helix, all residues except for the ones at the end, have their carbonyl oxygen hydrogen bonded to the amide nitrogens
• Alpha helix residue measurements

  • The rise per residue is 1.5 A (distance from one C alpha to the next)
  • Rotation per residue is 100 deg
  • 3.6 residues per rotation
• Beta sheets are polypeptide chains stacked. The strands can be parallel or antiparallel from each other.

  • Parallel: N and C terminus of each sheet match up
  • Antiparallel: N and C terminus of sheets don't match up
• On a strand in a beta sheet conformation, the carbonyls (and amides) will alternate directions allowing them to be interact with strands above and below them
• In the arrow representations of beta strands, the arrow points towards the carboxy-terminus
• Reverse turns: a secondary beta structure

  • These turns are secondary structure elements characterized by a sharp change in the direction of the polypeptide chain.
  • There is tighter H-bonding in these
• Fibrous proteins: alpha keratin

  Two alpha helices are intertwined and weak interactions like van der waals and ionic interactions hold them together.
• Myoglobin is a 153-residue that binds oxygen. 


  The iron atom in the heme group is critical for the binding of oxygen. The heme groups is a prosthetics group.
• Myoglobin is a soluble protein with hydrophobic residues packing in the interior and hydropillic residues at the surface. An exception are the two histidine residues that are important for binding the heme iron and oxygen
• Principles of tertiary structure

  Elements of secondary can come together to form supersecondary motifs. A motif often has a specific function.

  • the helix-turn-helix motif is a DNA binding motif. It is characterized by two alpha helices connected by a short loop, often forming a structure resembling a "bent" or "L-shaped" structure
  • The beta-hairpin is a structural motif consisting of two antiparallel beta strands connected by a reverse turn.
• Tertiary structures only have one polypeptide chain
• Principles of tertiary structure

  Some proteins fold into more than one compact regions known as domains. Domains are still a single polypeptide chain
• Quaternary structure

  some proteins consist of more than one polypeptide chain. Each chain forms a subunit
• Christian Anfinsen's experiment

  Meant to destroy the 3D structure of a protein and determine the conditions to restore its tertiary structure.

  • Results: when ribosome was treated with 2-mercaptoethanol in 8M urea, it became denatured. When denatured ribonuclease was freed of 2-mercaptoethanol and urea by dialysis, it regained activity.
  • Materials:
  • Ribonuclease: enzyme with 4 disulfide bonds
  • Urea: disrupts noncovalent bonds within proteins
  • 2-mercaptoethanol: an excess of it will cause cleavage of disulfide bonds by reducing the sulfides (SH) and oxidizing the mercaptoethanol
• significance of Anfinsen's experiment?

  Proves that the primary structure contains the information to specify a protein's 3D structure since a denatured protein (no 3D structure, mostly primary) can refold.
• Levinthal's paradox and folding

  With the amount of conformations a residue can adopt within a protein, its not possible for the proteins to fold in seconds whilst still sampling all possible conformations - and they don't.
• Folding funnel

  There isn't large of an energy difference between unfolded and folded states of a protein, so even proteins can easily move between different conformations.

  • Essentially, proteins fold by progressive stabilization rather than by pure chance.
  • Start at high entropy and energy where there are many possible conformations
  • molten globule states are where the protein may be partially folded but no compact structure yet
  • Partly correct intermediates are kept because they are still lower in energy until native structure is formed
• What type of proteins will not be depicted/applicable to the folding tunnel model?

  Intrinsically disordered proteins which only fold when there is a suitable binding partner present and do not rely on partially correct intermediates to guide folding
• Protein misfolding disease: prions

  Misfolded prion proteins can induce normal protein to change conformation, it is therefore infectious
• Ionic interactions- why can water dissolve salts?

  Ionic interactions involve complete charges, and water is able to shield charges due to its high dielectric constant, thus limiting interactions between the ions.
• Hydrophobic effect

  Non-polars aggregate together in a polar solvent.
• Why is the hydrophobic effect spontaneous?

  Water around a non-polar molecule is much more ordered than elsewhere in the solution, so by decreasing the amount of individual free floating non-polar molecules there are and having just one blob of non-polars, some of the water is released from more ordered positions
• Substituting uncharged/non-polar with polar amino acids (and vise versa) are the most disruptive changes
• Why is protein denaturation possible?

  Only hydrogen bonds hold the secondary structure together