Lecture 2 Paper 2

Protein chemistry

🧬 Big Picture: Why Site-Selective Protein Modification Matters

Nature constantly modifies proteins after translation using post-translational modifications (PTMs) such as phosphorylation, glycosylation, acetylation, methylation, and ubiquitination. These small chemical changes can:

  • Switch enzymes on or off
  • Alter protein–protein interactions
  • Change solubility, stability, and localization
  • Introduce new chemical functions not present in the 20 standard amino acids

💡 Core idea of the paper: Chemists aim to mimic nature by developing chemical reactions that modify proteins at one specific, pre-defined site, rather than randomly. This enables precision biology, imaging, and drug development.


❌ The Problem with Early Protein Modification Chemistry

Early methods (e.g. NHS esters) react with many lysines at once, producing:

  • Heterogeneous mixtures
  • Poor reproducibility
  • Difficult structure–function analysis
  • Reduced or lost biological activity

🚨 This is unacceptable for:

  • Mechanistic biology
  • Imaging probes
  • Therapeutic proteins

➡️ Solution: Site-selective protein-modification chemistry.


🎯 What Is Site-Selective Protein Modification?

Definition: A reaction that forms a covalent bond between a protein and a synthetic molecule at one specific residue.

Key requirements:

  • Chemoselective (one residue only)
  • Compatible with water, neutral pH, 20–37 °C
  • Does not disrupt protein folding
  • Stable linkage (especially in vivo)

🧪 Two Major Strategies

1️⃣ Natural amino acids (mostly Cys and N-terminus)

  • Works mainly in vitro
  • Requires purified proteins
  • Limited selectivity in complex mixtures

2️⃣ Non-canonical (unnatural) amino acids

  • Introduced via genetic code expansion
  • Carry unique handles (azide, alkyne, ketone, alkene, tetrazine)
  • Enable bioorthogonal chemistry
  • Work in cells and living organisms

🧠 Bioorthogonal Chemistry (Core Concept)

Bioorthogonal reactions:

  • Do not react with native cellular chemistry
  • Are fast, selective, and non-toxic
  • Allow protein-specific labeling inside cells or animals

Typical handles:

  • Azide ↔ alkyne (CuAAC)
  • Tetrazine ↔ strained alkene (IEDDA)
  • Ketone ↔ hydroxylamine (oxime ligation)

🧬 Studying Post-Translational Modifications (PTMs)

Why PTMs are hard to study:

  • Natural PTMs are often rare
  • Isolation of single modified isoforms is difficult
  • Enzymatic systems can be complex and context-dependent

🧩 Total and Semi-Synthesis Approaches

Native Chemical Ligation (NCL)

  • Joins peptide fragments via N-terminal Cys
  • Allows exact PTM placement
  • Excellent precision, poor scalability

Expressed Protein Ligation (EPL)

  • Combines synthetic peptides + recombinant proteins
  • Ideal when PTMs are near protein termini

📌 Classic example:

  • Histone H3 Arg42 dimethylation
  • Showed direct transcriptional activation

🧪 Chemical Installation of PTM Mimics on Folded Proteins

Instead of native PTMs, chemists install PTM mimics:

🔁 Cys → Dehydroalanine (Dha)

  • Cys chemically converted to Dha
  • Dha reacts with nucleophiles (Michael addition)

Examples:

  • Phosphorylation mimics (phospho-Cys)
  • Acetyl-Lys and methyl-Lys mimics
  • Functional histone analogues

💡 Key insight: Many mimics are biologically indistinguishable from native PTMs.


🧷 Ubiquitination by Chemical Ligation

Ubiquitination is especially challenging because it forms isopeptide bonds.

Chemical solutions include:

  • δ-thiol-Lys incorporation + NCL
  • Protected Lys strategies
  • Bioorthogonal polymerization of ubiquitin chains

🚀 Enables:

  • Site-specific mono- and poly-ubiquitination
  • Controlled studies of signaling pathways

🔬 Protein Modification for Imaging

Why site-selectivity matters:

  • In vivo behavior depends on label position
  • FRET requires precise dye placement
  • Double labeling demands orthogonal chemistry

🧪 In Vitro Labeling

  • Maleimides, haloacetamides, Michael acceptors
  • Mostly single-site, purified proteins

🧫 In Vivo & Live-Cell Labeling

Using bioorthogonal chemistry:

  • Proteins labeled inside living cells
  • Minimal background
  • High spatial and temporal precision

Examples:

  • Norbornene–tetrazine labeling on cell surfaces
  • “Turn-on” fluorophores (low background)
  • Tracking toxin uptake and trafficking

🧠 Protein-Based Biosensors

By combining:

  • Non-canonical amino acids
  • Environment-sensitive dyes
  • FRET pairs

Researchers created sensors for:

  • pH
  • Ca²⁺ concentration
  • Protein conformational changes

📍 Example:

  • Dual-labeled calmodulin reporting Ca²⁺ levels inside living cells

💊 Modifying Therapeutic Proteins

The problem:

  • Rapid clearance
  • Proteolysis
  • Immunogenicity
  • Loss of efficacy

🧴 PEGylation (and Its Limits)

PEGylation:

  • Increases hydrodynamic radius
  • Reduces kidney filtration
  • Shields from immune system

🚨 Traditional PEGylation:

  • Random
  • Often reduces activity

✅ Site-Selective PEGylation Strategies

  1. N-terminal oxidation → oxime ligation
  2. Direct N-terminal aldehyde chemistry
  3. Genetic encoding of ketone-bearing amino acids

📌 Clinical example:

  • Site-selective PEGylated human growth hormone
  • Improved pharmacokinetics
  • Reduced injection frequency

🎯 Antibody–Drug Conjugates (ADCs)

Why site-selectivity is critical:

  • Drug-to-antibody ratio (DAR) affects:
    • Stability
    • Toxicity
    • Clearance
  • Attachment site influences:
    • Efficacy
    • Off-target effects

🧪 Cys-Based ADC Chemistry

  • Engineered cysteines + maleimides
  • Can achieve DAR ≈ 2
  • But: maleimides can be unstable in plasma

🛠 Fixes:

  • Ring-opened maleimides
  • Neighboring amines
  • Alternative thiol-reactive chemistries

🧬 Bioorthogonal ADCs (Next-Gen)

  • Unnatural amino acids (e.g. p-acetyl-Phe)
  • Stable linkages
  • Homogeneous products
  • Improved in vivo performance

📈 Result:

  • Better tumor targeting
  • Reduced toxicity
  • Improved therapeutic index

🔓 In Situ Protein Activation (“Decaging”)

New frontier: activating proteins inside living cells

Strategies:

  • Palladium-mediated deprotection
  • Tetrazine-triggered Diels–Alder elimination
  • Photocaged amino acids (light-activated)

🔥 Applications:

  • Spatiotemporal control of protein function
  • Targeted pro-drug release
  • Precision therapeutics

🔮 Conclusions & Outlook

Two flavors of site-selective modification:

Natural amino acidsBioorthogonal chemistry
Simple, accessibleExtremely versatile
Mostly in vitroWorks in vivo
Limited positionsMultiple labels possible

🚀 The future:

  • Multi-site modifications
  • Live-cell functional studies
  • Safer, more effective protein drugs
  • Precision imaging and therapy

🧠 Key message: As bioorthogonal chemistry becomes easier and more accessible, site-selective protein modification will transform biology, imaging, and medicine.

Quiz

Score: 0/29 (0%)