Lecture 1 Book Supp

Protein chemistry

🧬 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.

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