Biology Chapter: Primary Structure of Proteins

Primary Structure of Proteins

• Proteins are unbranched polymers of amino acids (AAs) linked head to tail through covalent peptide bonds, forming an amide linkage.
• Peptide bond formation involves the release of water (H₂O) during the bond creation between the carboxyl group of one AA and the amino group of another.
• The peptide backbone consists of a repeated sequence of atoms: -N-Cα-Co.
  • N: Amide nitrogen
  • Cα: Alpha-carbon atom (chiral center, except for Gly)
  • Co: Carbonyl carbon
• The conformation of the peptide bond is energetically favored as the carbonyl oxygen and amide hydrogen are trans to each other, minimizing steric hindrance.
• The peptide bond exhibits partial double-bond character, resulting from resonance and contributing to rigidity in the polypeptide chain.
• Peptide bonds have approximately 40% double-bond character, preventing free rotation about the Co-N bond.

Polypeptide Backbone Properties

• The peptide backbone is relatively polar, with a protonated amide nitrogen and a negatively charged carbonyl oxygen creating a permanent dipole moment.
• The backbone's chemical reactivity is limited, with proton gain or loss occurring only at extreme pH levels.
• Peptides can be categorized based on the number of amino acids they comprise, such as dipeptides, tripeptides, tetrapeptides, and oligopeptides.

Steps for Protein Sequencing

• Step 1: Separation of Polypeptide Chains
  • Heteromultimer proteins are dissociated into their individual polypeptide chains through extreme pH, 8 M urea, 6 M guanidinium hydrochloride, or high salt concentrations.
• Step 2: Cleavage of Disulfide Bridges
  • Disulfide S-S bridges, which can link polypeptide chains, must be cleaved to isolate individual chains.
  • Performic acid oxidation converts disulfides into cysteic acids, preventing recombination due to electrostatic repulsion.
  • Reducing agents like 2-mercaptoethanol or dithiothreitol (DTT) can break S-S bonds, allowing formation of thiol (-SH) groups that may recombine later.
  • Alkylating agents (iodoacetate, 3-bromopropylamine) are employed after reduction to prevent new disulfide link formation.
• Step 3: N-terminal and C-terminal Residue Identification
  • The N-terminal and C-terminal residues are determined to help reconstruct the complete amino acid sequence.
• Step 4: Fragmenting and Sequencing
  • Polypeptide chains are cleaved into smaller fragments for analysis of amino acid composition and sequence.
  • Different cleavage techniques are applied repeatedly to generate overlapping sets of fragments for accurate sequence reconstruction.