Unit 4: Amino Acids and Proteins

I. Orientation

Amino acids are the monomeric units of proteins, while proteins are polymers whose biological properties depend on amino-acid sequence, three-dimensional folding, and chemical interactions. In aqueous cells near neutral pH, amino acids and proteins commonly exist as charged molecules, and their behavior is governed by acid–base equilibria, noncovalent forces, and peptide-bond formation.

• Common structural principle: Most protein amino acids are α-amino acids with an amino group, a carboxyl group, a hydrogen atom, and a variable side chain attached to the α-carbon.
• Biological convention: Protein sequences are written from the amino (N-) terminus to the carboxyl (C-) terminus.
• Chemical basis: Peptide bonds join amino acids by condensation, releasing water; hydrolysis reverses this process.
• Folding principle: The amino-acid sequence determines the possible three-dimensional conformations of a protein.
• Physiological condition: At approximately pH 7, acidic and basic groups may be ionized, so proteins often carry both positive and negative charges.

II. Amino Acids — Building Blocks and Acid–Base Behavior

Amino acids are organic compounds containing amino and carboxyl functional groups; their side chains determine their individual chemical and biological properties.

A. Structure and properties of amino acids

Structure and properties of amino acids explain how their common backbone and variable side chains produce different chemical behaviors.

• General structure: An α-amino acid has the formula:

      H
      |
H2N — C — COOH
      |
      R

• Symbols: R represents the variable side chain; C is the α-carbon to which the amino, carboxyl, hydrogen, and R groups are attached.
  • Zwitterion formation: In water, the amino group commonly accepts a proton and the carboxyl group loses one:

+H3N—CH(R)—COO−

• Meaning: This dipolar form has both a positive ammonium group and a negative carboxylate group but may have a net charge of zero.
  • Stereochemistry: Except glycine, amino acids are chiral because their α-carbon has four different substituents. Proteins contain predominantly L-amino acids.
  • Optical activity: L- and D-amino acids are mirror images; biological enzymes generally distinguish between them.
  • Peptide-bond reactivity: The carboxyl group of one amino acid reacts with the amino group of another, forming a peptide bond and releasing H₂O.
  • Side-chain effects: Nonpolar side chains favor hydrophobic environments, whereas polar or charged side chains interact with water, ions, and other biomolecules.
  • Special examples: Glycine (R = H) is flexible and achiral; proline has a ring connecting its side chain to the amino nitrogen and restricts backbone movement; cysteine contains a thiol group capable of disulfide-bond formation.

B. Classification of amino acids

Classification of amino acids organizes the twenty standard residues according to side-chain polarity, charge, structure, and nutritional requirement.

• Nonpolar aliphatic: Glycine, alanine, valine, leucine, isoleucine, methionine, and proline have largely hydrophobic side chains.
• Aromatic: Phenylalanine, tyrosine, and tryptophan contain aromatic rings; tyrosine is less hydrophobic because of its hydroxyl group.
• Polar uncharged: Serine, threonine, cysteine, asparagine, and glutamine can form hydrogen bonds without carrying a net charge near neutral pH.
• Acidic: Aspartate and glutamate contain additional carboxyl groups and are usually negatively charged near pH 7.
• Basic: Lysine, arginine, and histidine contain nitrogen atoms that can accept protons; lysine and arginine are generally positively charged, while histidine is often partly protonated near physiological pH.
• Essential versus nonessential: Essential amino acids, such as leucine and lysine, must be obtained sufficiently from the diet; nonessential amino acids can generally be synthesized by human metabolism.
• Conditionally essential: Some, such as arginine or glutamine, may be required from the diet during growth, illness, or metabolic stress.

C. Titration curves of amino acids

Titration curves of amino acids show how net charge changes as pH changes and identify the pH values at which ionizable groups lose protons.

• Acid–base groups: A simple amino acid has a carboxyl group with pKₐ₁ and an ammonium group with pKₐ₂.
• Henderson–Hasselbalch relationship:

pH = pKₐ + log([A−]/[HA])

• Symbols: [A−] is the concentration of deprotonated form; [HA] is the concentration of protonated form.
  • Titration sequence: At low pH, the amino acid is fully protonated with a net positive charge. As base is added, the carboxyl group loses a proton first, producing a zwitterion; at higher pH, the ammonium group loses its proton and the molecule becomes negatively charged.
  • Buffering regions: A relatively flat region near each pKₐ indicates resistance to pH change because protonated and deprotonated forms coexist.
  • Isoelectric point (pI): The pI is the pH at which the molecule has zero net charge. For amino acids without ionizable side chains:

pI = (pKₐ₁ + pKₐ₂)/2

• Worked example: If an amino acid has pKₐ₁ = 2.3 and pKₐ₂ = 9.6, then pI = (2.3 + 9.6)/2 = 5.95.
• Ionizable side chains: For aspartate, glutamate, lysine, arginine, histidine, cysteine, and tyrosine, the two pKₐ values surrounding the neutral species are used to calculate pI.

III. Proteins — Structure, Functions, and Organization

Proteins are biological macromolecules made of one or more polypeptide chains, and their functions arise from specific chemical structures and conformations.

A. Structure and functions of proteins

Structure and functions of proteins are linked because the sequence and folding of a polypeptide create binding sites, catalytic groups, and mechanical features.

• Polypeptide structure: Amino acids are connected by covalent peptide bonds between the carboxyl carbon of one residue and the amino nitrogen of the next.
• Peptide-bond properties: Resonance gives the peptide bond partial double-bond character, making it planar and restricting rotation.
• Catalysis: Enzymes such as amylase and pepsin accelerate reactions by positioning substrates and stabilizing transition states.
• Transport: Hemoglobin carries oxygen in red blood cells; membrane proteins transport ions and molecules across lipid bilayers.
• Structural support: Collagen provides tensile strength in connective tissue, while keratin strengthens hair, skin, and nails.
• Movement: Actin and myosin interact in muscle contraction; motor proteins also move cargo along cytoskeletal tracks.
• Signaling and regulation: Insulin regulates blood glucose, and protein receptors detect extracellular hormones or neurotransmitters.
• Defense: Antibodies recognize foreign molecules, while complement proteins and other immune proteins contribute to pathogen removal.
• Storage: Ferritin stores iron in a soluble protein shell; some proteins store amino acids or metal ions.

B. Different level of structural organization of proteins

Different level of structural organization of proteins describes the hierarchy from amino-acid sequence to complete functional assemblies.

• Primary structure: The primary structure is the linear amino-acid sequence linked by peptide bonds. A single substitution, such as valine replacing glutamate in hemoglobin β-chain position 6, can alter protein behavior.
• Secondary structure: Local backbone folding is stabilized mainly by hydrogen bonds between peptide carbonyl oxygen and amide hydrogen.
  • α-helix: A coiled structure in which hydrogen bonding commonly occurs between residues i and i + 4; side chains project outward.
  • β-sheet: Extended strands align in parallel or antiparallel arrangements, producing hydrogen-bonded sheets.
  • Turns and loops: These connect regular secondary structures and often occur at protein surfaces or active sites.
• Tertiary structure: The complete three-dimensional shape of one polypeptide results from hydrophobic interactions, hydrogen bonds, ionic interactions, van der Waals forces, and disulfide bonds.
  • Concrete example: A cysteine–cysteine disulfide bond forms by oxidation of two thiol groups, strengthening extracellular proteins such as insulin.
• Quaternary structure: Proteins containing multiple polypeptide subunits have quaternary structure. Hemoglobin, for example, contains two α and two β chains.
• Domains and motifs: A domain is a compact, independently folding region; a motif is a recurring structural pattern with a characteristic function.
• Structure–function relationship: Changes that disturb the active-site geometry, subunit arrangement, or binding surface can reduce or eliminate biological activity.

IV. Denaturation and Renaturation — Loss and Recovery of Protein Structure

Denaturation is the disruption of a protein’s native conformation without normally breaking its peptide backbone, whereas renaturation is recovery of the native structure when suitable conditions return.

A. Denaturation and renaturation of proteins

Denaturation and renaturation of proteins demonstrate that biological activity depends on higher-order structure and, in many cases, on the original amino-acid sequence.

• Denaturing agents: Heat, extreme pH, organic solvents, detergents, chaotropic agents such as urea, and heavy-metal ions can disrupt protein folding.
• Forces affected: Denaturation weakens hydrophobic interactions, hydrogen bonds, ionic interactions, or disulfide arrangements that maintain secondary, tertiary, and quaternary structure.
• What remains: Moderate denaturation usually does not hydrolyze peptide bonds, so the primary sequence remains intact.
• Observable effects: Denaturation may cause loss of enzyme activity, reduced solubility, aggregation, altered viscosity, or precipitation.
  • Example: Heating egg albumin changes a transparent soluble protein into an opaque solid because unfolded chains aggregate.
• Reversible denaturation: If the disrupting agent is removed before aggregation occurs, some proteins can refold and regain activity.
• Renaturation principle: Anfinsen’s experiments with ribonuclease showed that, under appropriate conditions, the amino-acid sequence can contain sufficient information for native folding.
• Limits of renaturation: Refolding may fail when aggregation, irreversible chemical modification, incorrect disulfide pairing, or cellular assistance requirements occur.
• Molecular chaperones: Chaperone proteins such as Hsp70 and chaperonins assist folding by preventing inappropriate interactions; they do not generally determine the final sequence-based structure.
• Reduction and oxidation: Reducing agents can break disulfide bonds, while controlled oxidation can restore them. Correct disulfide pairing is especially important for secreted proteins.
• Physiological significance: Misfolding and aggregation are associated with diseases including Alzheimer’s disease, Parkinson’s disease, and prion disorders; cells use quality-control systems to recognize and remove defective proteins.