🧬 Chapter 2 — Protein Composition and Structure

2.1 Proteins Are Built from a Repertoire of 20 Amino Acids

🔹 The amino acid alphabet

All proteins in all known life forms are built from the same 20 amino acids — a biochemical alphabet billions of years old. The extraordinary diversity of protein function comes not from new building blocks, but from different sequences and structures.

🔹 General amino acid structure

Each amino acid contains:

• An α-carbon (Cα)
• An amino group (–NH₃⁺)
• A carboxyl group (–COO⁻)
• A hydrogen
• A side chain (R group) → defines chemistry and function

All amino acids are chiral except glycine. Only L-amino acids are used in proteins.

Classification of Amino Acids

🟢 Hydrophobic (Nonpolar) Amino Acids

Examples: Gly, Ala, Val, Leu, Ile, Met, Pro, Phe, Trp

• Tend to avoid water
• Cluster in protein interiors
• Drive folding via the hydrophobic effect

Special cases:

• Glycine → tiny, flexible, fits into tight spaces
• Proline → cyclic, rigid, breaks helices
• Isoleucine & threonine → extra chiral center
• Tryptophan → bulky, aromatic, weakly polar

🔵 Polar, Uncharged Amino Acids

Examples: Ser, Thr, Tyr, Cys, Asn, Gln

• Participate in hydrogen bonding
• Often found on protein surfaces

Special:

• Cysteine → forms disulfide bonds
• Tyrosine → aromatic + polar (OH group)

🔴 Charged Amino Acids

• Positively charged: Lys, Arg, His
• Negatively charged: Asp, Glu

These residues:

• Form electrostatic interactions
• Are crucial for catalysis, binding, and pH sensitivity

2.2 Primary Structure: Amino Acid Sequence

🔹 What is primary structure?

The exact amino acid sequence of a protein.

🔹 Why sequence matters

The amino acid sequence:

1. Determines 3D structure
2. Determines function
3. Determines disease risk
4. Reveals evolutionary history

🧠 One amino acid change can cause disease Example:

• Sickle-cell anemia
• Cystic fibrosis

🔹 Disulfide bonds

• Formed by oxidation of two cysteine residues
• Create cystine
• Stabilize structure (especially extracellular proteins)

📌 Insulin is a classic example: two chains linked by disulfide bonds.

2.3 Secondary Structure: Local Folding Patterns

🔹 Peptide bond properties

• Planar
• Partial double-bond character
• Restricts rotation

🔹 Backbone angles

• ϕ (phi) and ψ (psi) angles define allowed conformations
• Visualized using Ramachandran plots

🌀 α-Helix

• Right-handed helix
• Stabilized by hydrogen bonds
• Side chains point outward
• Proline disrupts helices

Common in:

• Soluble proteins
• Membrane proteins (hydrophobic helices)

➿ β-Sheets

• Parallel or antiparallel strands
• Hydrogen bonds between strands
• Side chains alternate above and below the sheet

🔄 Turns & Loops

• Connect secondary structures
• Often contain glycine or proline
• Usually surface-exposed

Fibrous Proteins: Collagen

🧵 Collagen structure

• Triple helix
• Repeating Gly-X-Y motif
• X and Y often proline or hydroxyproline

📌 Every third residue must be glycine → Only glycine fits in the crowded center

🦴 Disease connection: Osteogenesis imperfecta

Replacing glycine with a larger residue:

• Disrupts folding
• Weakens collagen
• Causes brittle bones and blue sclera

2.4 Tertiary Structure: Overall Folding

🔹 Definition

The complete 3D structure of a single polypeptide chain.

🔹 Key principle

Water-soluble proteins fold into compact structures with hydrophobic cores

🧪 Myoglobin as a model protein

• 153 amino acids
• Mostly α-helical
• Contains heme prosthetic group
• Interior: hydrophobic residues
• Exterior: polar and charged residues

📌 Only two histidines inside — essential for oxygen binding.

2.5 Protein Folding and Stability

🔹 Folding is cooperative

Proteins fold via sharp transitions:

• Folded ↔ unfolded
• Few stable intermediates

This is an “all-or-none” process.

🔹 Why folding is not random

Random search would take longer than the age of the universe (Levinthal paradox).

Proteins fold by:

• Progressive stabilization of intermediates
• Energy funnel toward native state

⚠️ Misfolding and Aggregation

Some proteins misfold into amyloid fibrils:

• Rich in β-sheets
• Highly stable
• Insoluble

Diseases:

• Prion diseases
• Alzheimer’s
• Parkinson’s

Smaller oligomers may be more toxic than large aggregates.

2.6 Protein Modification and Cleavage

🔧 Post-translational modifications (PTMs)

Examples:

• Phosphorylation (Ser, Thr, Tyr)
• Hydroxylation (collagen)
• Acetylation
• γ-carboxylation

These:

• Regulate activity
• Enable signaling
• Add chemical functionality

✂️ Proteolytic processing

Proteins are often made as inactive precursors:

• Digestive enzymes
• Blood clotting factors
• Hormones
• Viral polyproteins

Cleavage activates or diversifies function.

✨ GFP (Green Fluorescent Protein)

• Fluorescence arises from Ser-Tyr-Gly rearrangement
• Spontaneous chemical modification
• Mutants span visible spectrum
• Essential biological marker

Chapter 2 Summary

Protein structure is hierarchical:

1. Primary → sequence
2. Secondary → helices, sheets
3. Tertiary → full fold
4. Quaternary → multi-subunit complexes

Structure → function → regulation → disease.

🔬 Chapter 3 — Exploring Proteins and Proteomes

3.1 Protein Purification

🔹 Why purify proteins?

To study:

• Structure
• Function
• Binding
• Catalysis

🧪 Purification strategies

Proteins differ in:

• Solubility
• Size
• Charge
• Binding specificity

Methods:

• Salting out
• Dialysis
• Gel-filtration chromatography
• Ion-exchange chromatography
• Affinity chromatography
• HPLC

📏 Gel filtration (size exclusion)

• Separates by size
• Larger proteins elute first
• Used to estimate molecular mass

⚡ Gel electrophoresis

• SDS-PAGE separates by mass
• SDS gives uniform negative charge
• Smaller proteins migrate faster

Advanced forms:

• Isoelectric focusing (pI separation)
• 2D electrophoresis (pI + size)

3.2 Immunological Techniques

🧬 Antibodies

• Bind specific epitopes
• Can be polyclonal or monoclonal

Used in:

• Western blotting
• ELISA
• Fluorescence microscopy

3.3 Mass Spectrometry

🔹 Core principle

Measures mass-to-charge ratio (m/z)

Ionization methods:

• MALDI
• ESI

Analyzers:

• Time-of-flight (TOF)

🔬 Tandem MS (MS/MS)

• Fragment peptides in predictable ways
• Determine sequence from fragment masses

Central to:

• Proteomics
• Complex protein mixtures
• Large assemblies

3.4 Peptide Sequencing

🧪 Edman degradation

• Labels N-terminal residue
• Removes one amino acid at a time
• Limited to short peptides

🧬 Overlap peptides

• Use multiple cleavage methods
• Reconstruct full sequence

Cleavage agents:

• Trypsin
• Chymotrypsin
• CNBr
• Carboxypeptidase

3.5 Peptide Synthesis

🔧 Solid-phase synthesis

• Automated
• C-terminus attached to resin
• Peptides used for:
  • Drugs
  • Antigens
  • Structural studies

3.6 Determining 3D Structure

🧊 X-ray crystallography

• Requires crystals
• Produces electron density maps
• Atomic resolution

📡 NMR spectroscopy

• Works in solution
• Uses chemical shifts
• Excellent for dynamics

Genomics vs Proteomics

DNA sequence:

• Predicts amino acid sequence

Protein analysis:

• Reveals modifications
• Reveals processing
• Reveals functional form

➡️ Both are complementary, not interchangeable.

Final Big Picture 🧠

Proteins:

• Use a simple alphabet
• Fold into complex structures
• Are regulated by chemistry, physics, and biology
• Can malfunction catastrophically

Understanding protein structure is essential to understanding life itself.