Unit 1: Amino Acids and Proteins

1. Amino Acids: Structure and Properties

Amino acids are the fundamental building blocks (monomers) of proteins. Each amino acid has a central carbon atom, called the alpha-carbon (α-carbon), which is bonded to four different groups:

• An amino group (—NH₂)
• A carboxyl group (—COOH)
• A hydrogen atom (—H)
• A variable side chain or R-group

General Structure of an Amino Acid. The R-group determines its properties.

Properties of Amino Acids

a) Zwitterions and Amphoteric Nature

At physiological pH (~7.4), the amino group is protonated (—NH₃⁺) and the carboxyl group is deprotonated (—COO⁻). This molecule with both positive and negative charges is called a zwitterion (German for "hybrid ion").

Because they can act as both an acid (donate H⁺) and a base (accept H⁺), amino acids are amphoteric.

b) Isoelectric Point (pI)

The isoelectric point (pI) is the specific pH at which an amino acid has no net electrical charge (i.e., it exists as a zwitterion). At a pH below its pI, it has a net positive charge. At a pH above its pI, it has a net negative charge.

c) Stereoisomerism

With the exception of glycine (whose R-group is just H), the α-carbon of all amino acids is chiral. This means they can exist as two non-superimposable mirror images: L-isomers and D-isomers. Biologically, all amino acids found in proteins are of the L-configuration.

Classification of Amino Acids (Based on R-Group)

There are 20 standard amino acids, which are classified by the properties of their R-groups:

2. Physical and Chemical Properties of Proteins

Proteins are polymers of amino acids linked by peptide bonds. Their properties are determined by their amino acid composition and 3D structure.

a) Physical Properties

• Solubility: Varies greatly. Globular proteins are generally soluble in water. Fibrous proteins are insoluble. Solubility is influenced by pH and salt concentration.
  • Salting In: At low salt concentrations, solubility increases.
  • Salting Out: At high salt concentrations, water molecules are competed for, and the protein precipitates. This is used in purification.
• Denaturation: The loss of a protein's native 3D structure (secondary, tertiary, and quaternary), leading to a loss of function. This is often irreversible.
  • Agents: Heat, extreme pH, organic solvents (e.g., alcohol), heavy metals, and detergents (e.g., SDS).
• Isoelectric Point (pI): Just like amino acids, proteins have a pI. At this pH, the protein has no net charge and is least soluble, often leading to precipitation.

b) Chemical Properties

• Peptide Bond Formation: Formed by a dehydration (condensation) reaction between the carboxyl group of one amino acid and the amino group of the next.
• Hydrolysis: Peptide bonds can be broken by hydrolysis (adding water), a reaction catalyzed by acids, bases, or enzymes (proteases).

3. Different Levels of Structural Organization of Proteins

This is a fundamental concept. Protein structure is described at four distinct levels.

a) Primary (1°) Structure

The primary structure is the unique, linear sequence of amino acids in a polypeptide chain, held together by peptide bonds.

This sequence is determined by the genetic code (DNA). Any change in the primary structure (a mutation) can alter the protein's function (e.g., sickle-cell anemia).

b) Secondary (2°) Structure

The secondary structure refers to the local, repetitive folding of the polypeptide backbone, stabilized by hydrogen bonds between the C=O and N-H groups of the backbone.

The two main types are:

• Alpha-Helix (α-helix): A right-handed coil. The H-bonds form between the C=O of one amino acid and the N-H of the amino acid four residues ahead. (e.g., in α-keratin).
• Beta-Pleated Sheet (β-sheet): Formed from two or more polypeptide segments lying side-by-side. The H-bonds form between the backbones of adjacent strands. Strands can be parallel or anti-parallel. (e.g., in silk fibroin).

c) Tertiary (3°) Structure

The tertiary structure is the overall three-dimensional folding of a single polypeptide chain, stabilized by interactions between the R-groups (side chains).

This structure defines the protein's specific biological function (e.g., the active site of an enzyme). Myoglobin is a classic example.

d) Quaternary (4°) Structure

The quaternary structure is the assembly of two or more separate polypeptide chains (subunits) into a single, functional protein complex. These subunits are held together by the same forces as tertiary structure.

Example: Hemoglobin, which consists of four subunits (two α-globin and two β-globin chains).

4. Forces Stabilizing Protein Structure and Shape

These forces are primarily responsible for the 3° and 4° structures.

1. Covalent Bonds:
   • Peptide Bonds: Stabilize primary structure.
   • Disulfide Bonds (—S—S—): A strong covalent bond formed between the sulfhydryl (—SH) groups of two cysteine residues. Crucial for stabilizing proteins like insulin and antibodies.
2. Non-Covalent Interactions (weaker, but collectively strong):
   • Hydrogen Bonds: Form between a hydrogen donor (like N-H or O-H) and a hydrogen acceptor (like C=O). Critical for α-helices, β-sheets, and tertiary folding.
   • Hydrophobic Interactions: This is the primary driving force for protein folding. Nonpolar (hydrophobic) R-groups cluster together in the protein's core, away from the surrounding water.
   • Electrostatic Interactions (Salt Bridges): Attractions between oppositely charged R-groups (e.g., between acidic Asp⁻ and basic Lys⁺).
   • Van der Waals Forces: Weak, short-range attractions between all atoms.

5. Fibrous and Globular Proteins

Proteins can be broadly classified based on their overall shape and function.

6. Protein Purification Techniques

To study a protein, it must be isolated from a complex mixture of cells. This involves two main stages: extraction and fractionation.

a) Protein Extraction

The first step is to get the proteins out of the cell. This is done by cell lysis (breaking open the cells) using methods like:

• Mechanical: Grinding, sonication (ultrasonic vibrations).
• Chemical: Detergents or enzymes (like lysozyme).

After lysis, cell debris is removed by centrifugation, leaving a crude extract containing the protein of interest.

b) Fractionation Techniques

These techniques separate proteins based on their unique physical properties (size, charge, binding affinity).

i. Salting Out

This technique separates proteins based on solubility. By adding a high concentration of a salt (like Ammonium Sulfate), the water molecules become solvated by the salt ions, reducing the water available to interact with the protein. The protein precipitates out. Different proteins precipitate at different salt concentrations.

ii. Chromatography

This is the most powerful set of purification techniques. A "mobile phase" (containing the protein mixture) is passed over a "stationary phase" (a column packed with a resin).

• Gel-Filtration Chromatography (Size-Exclusion):
  • Stationary Phase: Porous beads.
  • Separation Principle: Size. Large proteins cannot enter the beads and pass through the column quickly. Small proteins enter the beads, taking a longer, more complex path, and thus pass through slowly.
• Ion-Exchange Chromatography:
  • Stationary Phase: Charged beads (resin).
    • Anion-exchange: Positively-charged resin binds to negatively-charged proteins (anions).
    • Cation-exchange: Negatively-charged resin binds to positively-charged proteins (cations).
  • Separation Principle: Net charge. Proteins are eluted (released) by changing the pH or increasing the salt concentration.
• Affinity Chromatography:
  • Stationary Phase: Beads with a specific molecule (a ligand) covalently attached.
  • Separation Principle: Specific binding affinity. The protein of interest binds to the ligand. Other proteins wash through. The bound protein is then eluted by adding a solution that disrupts the binding. This is a very high-specificity technique.