Biochemistry of Peptides and Proteins

Peptides

• Polymers of amino acids
• Consist of two or three to thousands of linked amino acid residues
• Dipeptide: two amino acid molecules joined by a peptide bond

Condensation and Hydrolytic Reactions

• Dehydration reaction: removes a hydroxyl group and a hydrogen to form a covalent peptide bond
• Polypeptide backbone: repeating sequence of N-C-C-N-C-C… in the peptide bond
• Side chains (R groups) are not part of the backbone or the peptide bond
• Amino-terminal (N-terminal) residue: has a free -amino group
• Carboxyl-terminal (C-terminal) residue: has a free carboxyl group

Biologically Active Peptides

• Oxytocin: 9 amino acid residues (gly-leu-pro-cys-asn-gln-ile-tyr-cys)
• Glutathione: 3 amino acid residues (glu-cys-gly)
• Insulin: 51 amino acid residues
• Glucagon: 29 amino acid residues
• Adrenocorticotrophic hormone: 39 amino acid residues

Glutathione

• Tripeptide (glu-cys-gly)

Classification of Peptides

• Classified based on the number of amino acid residues:
  • Dipeptide: 2
  • Tripeptide: 3
  • Nanopeptide: 9
  • Oligopeptide: 10 to 20
  • Polypeptide: More than several dozens

Classification of Proteins

• Classified based on composition, shape, function, and solubility

Functions of Proteins

• Biological catalysts (enzymes)
• Hormones (insulin, glucagon)
• Neurotransmitters (glycine)
• Defense (antibodies)
• Transport in the body (albumin, hemoglobin)
• Locomotion (actin, myosin)
• Blood clotting (thrombin, fibrinogen)
• Structural (collagen, elastin, keratin)

Levels of Protein Organization

• Primary Structure: Amino acid sequence of the protein
• Secondary Structure: H bonds in the peptide chain backbone (alpha-helix and beta-sheets)
• Tertiary Structure: Non-covalent/covalent interactions between R groups within the protein
• Quaternary Structure: Interaction between 2 polypeptide chains

Primary Structure

• Unique sequence of amino acids
• Lysozyme: polypeptide chain of 129 amino acids
• Sickle cell disease: abnormal hemoglobins due to a single amino acid substitution
• Most abundant amino acids: Leu, Ala, Gly, Ser, Val, and Glu
• Rarest amino acids: Trp, Cys, Met, and His
• Amino acid sequence determines protein characteristics more than composition

Secondary Structure

• Results from hydrogen bonds along the polypeptide backbone
• Alpha-helix: coils
• Beta-pleated sheets: folds
• Silk fibers: strong due to many hydrogen bonds (beta pleated sheets)

Protein Folding: Secondary Structure

• Two regular folding patterns
  • Alpha-helix: protein turns like a spiral (found in fibrous proteins like hair, nails, and horns)
  • Beta-sheet: protein folds back on itself like a ribbon (found in globular proteins)

Features of Alpha-Helix

• Backbone tightly wound along an axis
• 3.6 amino acid residues per turn
• R groups project outwards
• Stabilized by hydrogen bonds between N-H and C=O groups (4 residues back)
• Each peptide bond participates in hydrogen bonding
• Most fibrous tissues have right-handed alpha-helices (more stable)
• Aromatic amino acids, charged, and hydrophobic amino acids can destabilize
• Glycine can cause coiling of the helix
• Proline terminates the helix by causing a kink

Features of Beta-Sheet

• Polypeptide chain fully extended
• 2-5 chains arranged side by side
• Can have chains running in the same direction (parallel beta-sheet)
• Can have chains running in opposite directions (anti-parallel beta-sheet)
• Stabilized by interchain hydrogen bonds
• Anti-parallel chains are more stable due to stronger hydrogen bonds

Tertiary Structure

• Determined by interactions among R groups and the polypeptide backbone
• Interactions include: hydrogen bonds, ionic bonds, hydrophobic interactions, van der Waals interactions

Quaternary Structure

• Aggregation of two or more polypeptide subunits
• Hemoglobin: globular protein with two copies of two kinds of polypeptides
• Lactate dehydrogenase, creatine phosphokinase

Protein Folding

• The peptide bond allows rotation and folding
• Weak non-covalent interactions (hydrogen bonds, electrostatic interactions, van der Waals forces) maintain the functional shape

Bonds Involved in Protein Folding

• Hydrophobic interactions
• Hydrogen bonds
• Electrostatic interactions (salt linkages)
• Van der Waals forces

Protein Conformation and Function

• Conformation gives a protein its unique function
• Proteins must interact with other molecules to work

Techniques for Separating and Purifying Proteins

• Dialysis
• Chromatography
  • Paper Chromatography
  • Thin-layer Chromatography (TLC)
  • Column Chromatography

Column Chromatography

• Stationary phase: small spherical beads coated with chemical functional groups
• Mobile phase: liquid percolated through the column
• Proteins interact based on charge, hydrophobicity, or ligand-binding properties

Partition Chromatography

• Separation based on relative affinity of proteins for the stationary and mobile phases
• Proteins with a stronger affinity for the stationary phase are retained longer
• Retention time is affected by the composition of both phases
• Retardation factor (Rf) can be calculated for an amino acid/protein in a solvent.

Size Exclusion Chromatography

• Separates proteins based on size (molecular mass and shape)
• Also known as Gel Filtration
• Proteins too large to enter the pores are excluded and emerge first
• Proteins emerge in descending order of size

Absorption Chromatography

• Protein mixture is applied under conditions where the target protein strongly associates with the stationary phase
• Non-adhering molecules are eluted first and discarded
• Proteins are sequentially released by altering the forces that stabilize the protein-stationary phase complex (using a gradient of increasing salt concentration or changing pH)

Ion Exchange Chromatography

• Charged molecules bind to oppositely charged groups on a matrix
• Anions bind to cationic groups (anion exchangers)
• Cations bind to anionic groups (cation exchangers)
• DEAE (diethylaminoethyl) groups: anion exchanger
• CM (carboxymethyl) groups: cation exchanger
• Binding affinity depends on competing ions and the pH
• Proteins with a net negative charge adhere to positively charged beads (anion exchangers)
• Proteins compete with monovalent ions for binding
• Bound proteins are displaced by increasing the concentration of monovalent ions in the mobile phase
• Changing pH can achieve sequential elution

Electrophoresis

• Movement of ions in an electrical field
• Positively charged amino acids move towards the cathode
• Negatively charged amino acids move towards the anode
• Uncharged amino acids remain at the point of application

Importance of Protein Sequencing

• Confirms effects of mutation
• Key in vaccine synthesis (knowledge of proteins on viral membranes)
• Explains mechanisms of enzyme catalysis
• Key in DNA cloning

This quiz covers the structure and classification of peptides, including dipeptides and polypeptides. Learn about biologically active peptides and their functions, as well as the biochemical reactions involved in their formation and breakdown. Test your knowledge of amino acid residues and peptide bonds with this comprehensive assessment.