Proteins

Chapter 1: Introduction to Proteins

Proteins are macromolecules found in every living organism. They are essential for nutrition, structure, and the regulation of chemical processes that sustain life. The term "protein" was coined in 1838 by Swedish chemist Jöns Jacob Berzelius, derived from the Greek word *proteios*, meaning "of first importance" or "most important."

A key feature of proteins is their **specificity**. Proteins differ between species. Even within a single organism, proteins in muscle differ from those in the brain, liver, or blood. This specificity arises from differences in the sequence of their building blocks.

## Chapter 2: The Building Blocks – Amino Acids

All proteins are polymers of **α-amino acids**. An α-amino acid consists of a central carbon (the α-carbon) bonded to four groups:

1. An amino group (–NH₂)

2. A carboxyl group (–COOH)

3. A hydrogen atom (–H)

4. A variable side chain (R group)

### 2.1 General Properties of Amino Acids

- **Zwitterion formation:** In aqueous solution (pH 4–8), the amino group gains a proton (–NH₃⁺) and the carboxyl group loses one (–COO⁻). This dipolar ion is called a **zwitterion**. At this pH, the molecule has no net charge and does not migrate in an electric field.

- **Acidic conditions (pH < 4):** –COO⁻ converts to –COOH (uncharged).

- **Alkaline conditions (pH > 9):** –NH₃⁺ loses a proton to become –NH₂.

- **Optical activity:** Except for glycine (R = H), all amino acids have an asymmetric α-carbon and exist as mirror-image isomers (L and D forms). Proteins almost exclusively contain **L-amino acids**. (D-amino acids occur only in some bacterial peptides like gramicidin.)

### 2.2 The 20 Common Amino Acids

Although over 100 amino acids exist in nature, only 20 are commonly found in proteins. They are grouped by their side chains (R groups).

#### Group 1: Nonpolar (Hydrophobic) Amino Acids

These have no affinity for water but attract each other. Animals can synthesize only alanine and glycine; the rest are essential in the diet.

- **Glycine (Gly, G):** R = H (simplest, no asymmetry)

- **Alanine (Ala, A):** R = –CH₃

- **Valine (Val, V):** R = –CH(CH₃)₂

- **Leucine (Leu, L):** R = –CH₂–CH(CH₃)₂

- **Isoleucine (Ile, I):** R = –CH(CH₃)–CH₂CH₃

- **Proline (Pro, P):** Unique – the side chain loops back to bond with the amino group, forming a ring. This creates "kinks" in peptide chains. Cannot form a zwitterion.

#### Group 2: Polar (Hydrophilic) Uncharged Amino Acids

- **Serine (Ser, S):** R = –CH₂OH (contains alcohol group)

- **Threonine (Thr, T):** R = –CH(OH)CH₃

- **Cysteine (Cys, C):** R = –CH₂SH (contains mercapto group). Easily oxidized to **cystine** (two cysteines linked by a disulfide bond –S–S–). Disulfide bonds are critical for protein folding.

- **Asparagine (Asn, N):** R = –CH₂CONH₂

- **Glutamine (Gln, Q):** R = –CH₂CH₂CONH₂

#### Group 3: Acidic (Negatively Charged)

- **Aspartic acid (Asp, D):** R = –CH₂COOH

- **Glutamic acid (Glu, E):** R = –CH₂CH₂COOH

These are dicarboxylic acids. At physiological pH, their side chains are –COO⁻.

#### Group 4: Basic (Positively Charged)

- **Lysine (Lys, K):** R = –(CH₂)₄NH₃⁺ (synthesized by plants, not animals)

- **Arginine (Arg, R):** R = guanidinium group (strongly basic; abundant in protamines)

- **Histidine (His, H):** R = imidazole ring (weak base; acts as a buffer at pH 7)

#### Group 5: Aromatic (Absorb UV Light)

These contain a benzene ring. All are essential amino acids. They absorb UV light at 270–290 nm, allowing protein quantification.

- **Phenylalanine (Phe, F):** R = –CH₂C₆H₅ (weak UV absorption)

- **Tyrosine (Tyr, Y):** R = –CH₂C₆H₄OH (strong UV absorption)

- **Tryptophan (Trp, W):** R = indole ring (strongest UV absorption)

### 2.3 Uncommon Amino Acids

Some proteins contain modified amino acids in small amounts (<2%):

- **Hydroxyproline** and **hydroxylysine** in collagen.

- **Monomethyl-, dimethyl-, trimethyllysine** in some proteins.

## Chapter 3: Peptide Bonds and Protein Chain Formation

Amino acids link via **covalent peptide bonds**. The carboxyl group of one amino acid reacts with the amino group of the next, releasing a water molecule (condensation reaction).

- **Dipeptide:** 2 amino acids

- **Tripeptide:** 3 amino acids

- **Polypeptide:** Many amino acids (typically >100)

- **Protein:** A functional polypeptide or complex of polypeptides.

**Conventions:** Write peptides with the free α-amino group (N-terminus) on the left and the free carboxyl group (C-terminus) on the right.

**Molecular weight:** Average amino acid residue weight = ~110 daltons. Most proteins range from 10,000 to 100,000 daltons. (One dalton = mass of one hydrogen atom).

## Chapter 4: Levels of Protein Structure

### 4.1 Primary Structure

The linear sequence of amino acids. This determines all higher levels of structure. Even a small change can cause disease (e.g., sickle cell anemia: glutamate → valine in hemoglobin).

**Determining primary structure (Edman degradation):**

1. React the N-terminal amino acid with phenyl isothiocyanate.

2. Cleave it off without breaking other bonds.

3. Identify the amino acid.

4. Repeat.

Limitation: Only works for 30–50 residues. Therefore, proteins are first cut into smaller peptides using enzymes like **trypsin** (cleaves after lysine/arginine) or **chymotrypsin** (cleaves after tyrosine/phenylalanine/tryptophan). Overlapping fragments are used to reconstruct the full sequence.

### 4.2 Secondary Structure

Local, regular folding patterns stabilized by **hydrogen bonds** between the backbone –NH and –C=O groups.

- **α-Helix:** Right-handed spiral. 3.7 amino acids per turn, pitch = 5.4 Å. Stabilized by hydrogen bonds between every fourth amino acid. Found in myoglobin and many other proteins.

- **β-Pleated Sheet:** Extended, zigzag chains aligned side by side. Can be **parallel** (chains run same direction) or **antiparallel** (opposite directions). Hydrogen bonds form between adjacent chains. Found in silk fibroin.

### 4.3 Tertiary Structure

The three-dimensional folding of a single polypeptide chain, including side chains. Stabilized by:

- **Hydrogen bonds** (between polar side chains)

- **Hydrophobic interactions** (nonpolar side chains cluster away from water)

- **Salt bridges** (ionic bonds between acidic and basic side chains, e.g., glutamate with lysine)

- **Disulfide bonds** (covalent S–S bonds between cysteines)

- **Metal ion coordination**

Globular proteins (e.g., hemoglobin, enzymes) have tightly folded tertiary structures. Fibrous proteins (e.g., collagen, keratin) have elongated tertiary structures.

### 4.4 Quaternary Structure

The arrangement of multiple polypeptide subunits into one functional protein. Subunits are held together by noncovalent bonds (hydrogen bonds, hydrophobic interactions) and sometimes disulfide bonds.

**Examples:**

- **Hemoglobin:** Tetramer of 2α and 2β chains (molecular weight ~64,500)

- **Immunoglobulin G (IgG):** Four chains (2 heavy, 2 light)

- **Collagen:** Triple helix of three polypeptide chains

## Chapter 5: Important Physicochemical Properties

### 5.1 Hydration and Solubility

Proteins bind water (10–20% of their weight) via charged and polar side chains. Globular proteins are generally water-soluble. Fibrous proteins are insoluble. Adding salts like ammonium sulfate **salts out** (precipitates) proteins by competing for water.

### 5.2 Denaturation

Loss of tertiary/quaternary structure (not primary structure) due to:

- Heat (boiling an egg – irreversible)

- Strong acids or bases

- Organic solvents (ethanol, acetone)

- Urea or guanidinium chloride (break hydrogen bonds)

- Reducing agents (break disulfide bonds)

Denatured proteins lose biological activity and often become insoluble. **Renaturation** (refolding) is possible for some small proteins (e.g., ribonuclease).

### 5.3 Isoelectric Point (pI)

The pH at which a protein has no net charge and does not migrate in an electric field.

- Most proteins: pI 5–7

- Pepsin (very acidic): pI ~1

- Protamines (very basic): pI 11–12

### 5.4 Spectrophotometric Properties

Proteins absorb UV light at 280 nm due to tyrosine and tryptophan. This is used to measure protein concentration.

## Chapter 6: Classification of Proteins

### 6.1 Classification by Solubility (Traditional)

| Class | Solubility | Examples |

|-------|------------|----------|

| Albumins | Water, dilute salt | Serum albumin, ovalbumin |

| Globulins | Insoluble in water, soluble in salt solutions | Immunoglobulins, edestin |

| Prolamins | 50–80% ethanol | Gliadin (wheat) |

| Glutelins | Dilute acid or alkali | Glutenin |

| Protamines | Very basic, soluble in water | Salmine (salmon sperm) |

| Histones | Basic, soluble in water | Nuclear histones |

| Scleroproteins | Insoluble | Collagen, keratin, elastin, fibroin (silk) |

### 6.2 Classification by Composition (Conjugated Proteins)

| Type | Prosthetic Group | Example |

|------|----------------|---------|

| Glycoproteins | Carbohydrates | Mucins, immunoglobulins, orosomucoid |

| Lipoproteins | Lipids | LDL, HDL, VLDL |

| Phosphoproteins | Phosphate (on serine) | Casein (milk), phosvitin (egg yolk) |

| Hemoproteins | Heme (iron-porphyrin) | Hemoglobin, myoglobin, cytochromes |

| Metalloproteins | Metal ions | Ferritin (Fe), Ceruloplasmin (Cu), Transferrin (Fe) |

| Nucleoproteins | Nucleic acid (DNA or RNA) | Chromosomes, ribosomes, viruses (TMV) |

| Chromoproteins | Pigments | Melanin, phycoerythrin, biliproteins |

### 6.3 Classification by Biological Function (Modern)

| Function | Description | Examples |

|----------|-------------|----------|

| Structural | Provide shape and support | Collagen, keratin, elastin |

| Contractile | Enable movement | Myosin, actin |

| Transport | Carry small molecules | Hemoglobin (O₂), Albumin (fatty acids), Transferrin (Fe) |

| Catalytic | Speed up chemical reactions | All enzymes |

| Hormonal | Regulate metabolism | Insulin, growth hormone, glucagon, thyrotropin |

| Protective | Defend against pathogens | Immunoglobulins (antibodies), thrombin (clotting) |

| Storage | Reserve amino acids or minerals | Casein (milk), ferritin (iron), ovalbumin (egg) |

| Receptor | Receive signals | Cell surface receptors |

## Chapter 7: Detailed Examples of Major Proteins

### 7.1 Hemoglobin and Myoglobin

- **Hemoglobin:** Tetramer (α₂β₂), molecular weight = 64,500. Oxygen carrier in blood. Cooperativity: binding of one O₂ increases affinity for the next.

- **Myoglobin:** Monomer, MW = 16,500. Oxygen storage in muscle.

- **Fetal hemoglobin (HbF):** Has γ chains instead of β; higher oxygen affinity.

- **Sickle cell anemia:** Single amino acid substitution (glutamic acid → valine at position 6 of β-chain) causes polymerization and deformed red blood cells.

- **Methemoglobin:** Iron in Fe³⁺ state (cannot bind O₂).

- **Carbon monoxide poisoning:** CO binds to heme with 200× greater affinity than O₂.

### 7.2 Collagen

- Most abundant protein in mammals (25–35% of total protein).

- Found in skin, bone, tendon, cartilage, ligaments.

- Contains **hydroxyproline** and **hydroxylysine** (modified after synthesis).

- Triple helix of three polypeptide chains (each ~1000 amino acids).

- High glycine content (every third residue is glycine).

- Proline creates kinks, preventing α-helix formation.

- Boiling converts collagen to **gelatin** (chains separate and trap water).

- Tanning (chromium salts) cross-links collagen fibers to make leather.

- Vitamin C deficiency (scurvy) impairs collagen synthesis.

### 7.3 Keratin

- Found in hair, nails, wool, feathers, skin.

- High cystine content (up to 24% of residues) → many disulfide bonds → very stable.

- Insoluble and resistant to proteolytic enzymes.

- **Permanent hair waving:** Reduce disulfide bonds (thioglycolate), reshape, re-oxidize (air or H₂O₂).

### 7.4 Immunoglobulins (Antibodies)

- **Structure:** Y-shaped tetramer of 2 heavy (H) chains (MW ~55,000 each) and 2 light (L) chains (MW ~22,000 each). Disulfide bonds hold them together.

- **Fragments:** Papain cleaves IgG into 2 Fab (antigen-binding) fragments and 1 Fc (crystallizable) fragment.

- **Classes:** IgG (MW 150,000, most abundant), IgA (dimer, in secretions), IgM (pentamer, first response), IgD, IgE.

- **Bence-Jones proteins:** Light chains found in urine of multiple myeloma patients.

- **Allotypes:** Genetic variations in constant regions (e.g., InV on κ chains, Gm on γ chains).

### 7.5 Muscle Proteins

- **Myosin:** MW ~500,000. Elongated molecule with globular heads. Has ATPase activity. Constitutes ~55% of muscle protein.

- **Actin:** Globular (G-actin) polymerizes to fibrous (F-actin). MW ~50,000.

- **Actomyosin:** Complex of myosin and actin responsible for contraction.

- **Tropomyosin:** Smaller, rod-shaped, regulates contraction.

- **Mechanism:** Sliding filament model – myosin heads pull actin filaments toward center.

### 7.6 Fibrinogen and Fibrin (Clotting)

- **Fibrinogen:** Soluble plasma protein (MW 340,000). Three pairs of chains. Rod-shaped.

- **Thrombin** (enzyme) cleaves off fibrinopeptides A and B → fibrin monomer.

- Fibrin monomers polymerize noncovalently, then factor XIII cross-links via new peptide bonds → stable clot.

## Chapter 8: Enzymes (Catalytic Proteins)

Enzymes are proteins that accelerate chemical reactions without being consumed. They are highly specific. Most enzymes have molecular weights between 20,000 and 100,000.

### 8.1 Classification (Six Main Classes)

| Class | Reaction Type | Example |

|-------|---------------|---------|

| 1. Oxidoreductases | Transfer electrons (oxidation/reduction) | Alcohol dehydrogenase |

| 2. Transferases | Transfer functional groups | Pyruvate kinase |

| 3. Hydrolases | Hydrolysis (add water) | Trypsin, pepsin |

| 4. Lyases | Remove/add groups without water | Aldolase |

| 5. Isomerases | Rearrange atoms | Phosphoglucose isomerase |

| 6. Ligases | Join molecules using ATP | DNA ligase |

### 8.2 Cofactors, Coenzymes, and Prosthetic Groups

Many enzymes require non-protein components:

- **Cofactor:** General term (metal ions like Mg²⁺, Zn²⁺, Fe²⁺).

- **Prosthetic group:** Tightly bound organic molecule (e.g., heme in cytochromes).

- **Coenzyme:** Loosely bound organic molecule that shuttles chemical groups (e.g., NAD⁺, ATP, coenzyme A).

**Holoenzyme** = Apoenzyme (protein part) + Cofactor.

**Example:** Nicotinamide adenine dinucleotide (NAD⁺) carries hydrogen atoms in redox reactions.

### 8.3 Mechanism of Action

- **Active site:** Specific region where substrate binds.

- **Lock-and-key model:** Substrate fits exactly into rigid active site (Emil Fischer, 1899).

- **Induced-fit model:** Substrate binding induces conformational change in enzyme (more widely accepted today).

- **Transition state stabilization:** Enzymes lower activation energy.

- **Turnover number:** Molecules of product per enzyme per second (e.g., catalase: 10⁷ per second).

### 8.4 Enzyme Kinetics (Michaelis-Menten)

- **Vmax:** Maximum velocity when all active sites are saturated.

- **Km** (Michaelis constant): Substrate concentration at half Vmax. Inverse measure of substrate affinity.

- **Lineweaver-Burk plot:** Double reciprocal plot (1/V vs. 1/[S]) gives straight line.

### 8.5 Enzyme Inhibition

|------|---------------|--------------|---------|

### 8.6 Allosteric Regulation

- **Allosteric sites:** Regulatory sites separate from the active site.

- **Allosteric activators** increase activity; **inhibitors** decrease activity.

- **Cooperativity:** Binding of one substrate molecule affects binding of subsequent ones.

- Positive cooperativity: Easier subsequent binding (hemoglobin, O₂) → sigmoidal curve.

- Negative cooperativity: Harder subsequent binding.

- **Feedback inhibition:** End product of a pathway inhibits the first enzyme (e.g., histidine inhibits first enzyme in its own synthesis).

### 8.7 Examples of Important Enzymes

| Enzyme | Substrate | Function |

|--------|-----------|----------|

| Pepsin | Proteins (protease) | Digestion in stomach (pH ~2) |

| Trypsin | Proteins (cleaves after Lys/Arg) | Digestion in small intestine |

| Chymotrypsin | Proteins (cleaves after aromatic) | Digestion |

| Ribonuclease | RNA | Breaks down RNA |

| DNA polymerase | DNA building blocks | DNA replication |

| Catalase | H₂O₂ | Breaks down hydrogen peroxide |

| Acetylcholinesterase | Acetylcholine | Terminates nerve impulses |

| Urease | Urea | First enzyme crystallized (1926) |

## Chapter 9: Protein Hormones

Hormones are chemical messengers. Many are peptides or proteins (others are steroids).

|---------|--------|-----|----------|

**Proinsulin:** Single-chain precursor (MW ~9,000). Removal of C-peptide (connecting peptide, ~33 aa) yields active two-chain insulin.

## Chapter 10: Plant Proteins

| Protein | Source | Characteristics |

|---------|--------|----------------|

| Edestin | Hemp seed | Globulin, hexamer (MW 310,000) |

| Amandin | Almonds | MW 330,000 |

| Concanavalin A | Jack bean | Lectin (binds sugars) |

| Gliadin | Wheat | Prolamin, rich in proline and glutamine, low in lysine |

| Glutenin | Wheat | Glutelin, gives dough elasticity |

| Zein | Corn | Prolamin, low in lysine and tryptophan |

| Leghemoglobin | Root nodules (legumes) | Red heme protein, binds oxygen, involved in nitrogen fixation |

**Nutritional note:** Most plant proteins are deficient in one or more essential amino acids (e.g., cereals lack lysine; legumes lack methionine). Combining cereals with legumes provides a complete protein.

## Chapter 11: Protein Methods (Summary)

### Determining Quantity

- **Kjeldahl method:** Measure total nitrogen; protein = N × 6.25 (assuming 16% N).

- **Biuret reaction:** Violet color with Cu²⁺ in alkaline solution.

- **UV absorbance at 280 nm** (tyrosine/tryptophan content).

- **Lowry method** and **Bradford assay** (dye-binding).

### Determining Purity

- **Electrophoresis (SDS-PAGE):** Separates by molecular weight.

- **Isoelectric focusing:** Separates by pI.

- **Ultracentrifugation:** Sedimentation velocity gives molecular weight and shape.

- **Gel filtration (size exclusion):** Separates by size.

- **Ion exchange chromatography:** Separates by charge.

### Determining Sequence

- **Edman degradation** (for N-terminal sequence).

- **Mass spectrometry** (modern method; can sequence entire proteins).

## Chapter 12: Protein Nutrition

### Essential Amino Acids (Humans cannot synthesize)

Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, Valine.

### Nitrogen Balance

- **Positive balance:** Growth, pregnancy, recovery from illness (intake > excretion).

- **Negative balance:** Starvation, severe illness, low-protein diet (excretion > intake).

- **Minimum daily requirement:** ~0.8 g protein per kg body weight for adults.

### Protein Quality

- **Complete proteins:** Contain all essential amino acids (animal sources: meat, fish, eggs, milk, cheese; also soy and quinoa).

- **Incomplete proteins:** Lack one or more essential amino acids (most plant foods).

- **Protein complementation:** Combining rice (low lysine, high methionine) with beans (high lysine, low methionine) provides a complete profile.

## Chapter 13: Protein Diseases and Disorders

| Condition | Defect |

| Sickle cell anemia | Hemoglobin S (Glu→Val in β-chain) |

| Phenylketonuria (PKU) | Lack phenylalanine hydroxylase → phenylalanine accumulation |

| Albinism | Lack tyrosinase → no melanin |

| Galactosemia | Lack galactose-1-phosphate uridyl transferase |

| Cystinuria | Defective kidney transport of cysteine |

| Marfan syndrome | Defective fibrillin (connective tissue protein) |

| Osteogenesis imperfecta | Defective collagen type I (brittle bones) |

| Scurvy | Vitamin C deficiency → impaired collagen hydroxylation |

| Kwashiorkor | Severe protein deficiency (edema, fatty liver, growth failure) |

| Multiple myeloma | Malignant plasma cells produce excess monoclonal immunoglobulin or Bence-Jones protein |

Conclusion

Proteins are the most diverse and functionally important macromolecules in living systems. Their structure spans four hierarchical levels (primary to quaternary), each determined by the sequence of just 20 standard amino acids. From enzymes that catalyze life's reactions to antibodies that defend against infection, from hemoglobin that transports oxygen to collagen that holds the body together, proteins perform an extraordinary range of tasks. The relationship between structure and function is absolute: any alteration in primary structure can lead to loss of function or disease. Understanding proteins is therefore fundamental to biochemistry, medicine, nutrition, and biotechnology.

Proteins

Discussion