🧩 Resonance Assignment – Homonuclear Approach (Part 1)

🎯 What is Resonance Assignment?

Resonance assignment is the process of determining:

• Which NMR signal belongs to which atom
• Or ideally: knowing the chemical shift of every atom in the protein

In practice:

• You rarely get 100%
• But >95% assignment is possible

This is essential before you can interpret structure, dynamics, or interactions.

🛠 Manual vs Automatic Assignment

There are two general approaches:

🧑‍🔬 Manual Assignment

You do it yourself by analyzing spectra and connecting signals.

🤖 Automated Assignment

Software performs the same logic as humans — but:

• Only works well if peak lists are clean
• Requires curated, high-quality data
• Still follows the same principles humans use

This lecture focuses on the manual approach.

🔬 Two Assignment Strategies (Depends on System)

This lecture covers:

🧬 Homonuclear assignment using the Sequential Walk

🧠 The Key Concept: Spin Systems

What is a spin system?

A spin system is:

A group of nuclei connected by scalar (J) coupling

In homonuclear ¹H NMR:

• Each amino acid forms its own independent spin system

Why?

Because:

• Coupling across the peptide bond would require 4-bond coupling
• 4-bond ¹H-¹H coupling is too weak to observe
• Therefore: no scalar coupling between different residues

👉 Each amino acid = isolated proton network

💧 Why Can We See NH Protons?

The spectra are recorded in normal water (H₂O), not D₂O.

Amide NH:

• Is in resonance with carbonyl
• Has slow exchange with water
• Therefore visible
• Shows 3-bond coupling to Hα

So NH signals are observable and useful for assignment.

📊 Required Spectra

To perform sequential walk in homonuclear NMR, you need:

• COSY
• TOCSY
• NOESY

(If unfamiliar with these, the video suggests reviewing them first.)

🔎 Chemical Shift Regions

Approximate ¹H shifts:

In 2D homonuclear spectra:

• Same chemical shift ranges appear on both axes

📘 COSY vs TOCSY – What Do They Show?

🔷 COSY

Shows:

• Direct scalar coupling (mostly 3-bond)

You expect:

• NH ↔ Hα cross peaks
• Hα ↔ Hβ
• Hβ ↔ Hβ (sometimes poorly resolved)

So in COSY: You see pairwise couplings only.

🔷 TOCSY

Shows:

• All protons connected by uninterrupted coupling networks

This means:

• From NH, you can potentially see:
  • Hα
  • Hβ
  • Hγ
  • Hδ
  • etc.

Signal intensity:

• Decreases further away from NH
• May not see full side chain

🔗 When Does TOCSY NOT Show Full Side Chain?

Two important exceptions:

1️⃣ Methionine

Chain: NH → Hα → Hβ → Hγ → S → Hε

The sulfur breaks coupling. NH to methyl beyond sulfur = 4-bond coupling → not observed.

2️⃣ Aromatic residues

No coupling between:

• Aliphatic backbone
• Aromatic ring system

So full chain not always visible.

🔢 Step 1: Identify Spin Systems

You:

1. Pick NH peaks
2. Follow vertical TOCSY strips
3. Assign all coupled protons
4. Label spin systems arbitrarily (1, 2, 3, 4…)

Now you have isolated amino-acid-like units.

🧬 Recognizing Amino Acid Types from Spin Systems

This is pattern recognition.

🟢 Glycine

• Two Hα
• Nothing else

🟡 Alanine

• One Hα
• One methyl group

🟠 Threonine

• One Hα
• One methyl
• Hβ unusually high shift (bound to oxygen)
• Hβ can be higher than Hα

Only amino acid where this happens.

🔵 Valine

• One Hβ
• Two methyl groups

Distinctive pattern.

🟣 AMX Spin Systems

Group with:

• One Hα
• Two Hβ
• Possibly more protons

Includes:

• Serine
• Aspartate
• Asparagine
• Cysteine
• Aromatics

Harder to distinguish.

🟤 Leucine & Isoleucine

• Many methyl groups
• Larger spin systems
• Often distinguishable from pattern

🔴 Lysine & Arginine

• Long side chains
• Final side-chain CH₂ around 3–3.5 ppm
• Unique signature

⚫ Proline

Invisible in this method.

Why?

• No NH (secondary amine in peptide bond)
• So no starting point

📊 Sanity Check: Counting Spin Systems

After identifying spin systems:

Ask:

• Does number roughly match expected residues?
• Expected number:
  Total amino acids – number of prolines – possibly 1(N - terminus)

Why N-terminus may be invisible:

• It is an amine, not amide
• Exchanges too fast with water

If:

• 30 residues → found 12 → problem
• 12 residues → found 30 → serious problem

Small deviations are fine (overlap, weak peaks).

🧩 What Do We Have After This?

You now know:

• Individual spin systems
• Probable amino acid types
• But NOT:
  • Their order
  • Their position in sequence

Example from lecture:

• Alanine
• Leucine/Isoleucine
• AMX residue
• Alanine

But which alanine is which?

🚶 The Sequential Walk (Preview)

The next step (Part 2) will:

• Connect spin systems
• Use NOESY
• Establish sequential relationships
• Achieve sequence-specific assignment

That’s where:

Spin system → specific residue number

🧠 Core Concepts to Remember

• Each residue = independent spin system
• COSY → direct couplings
• TOCSY → full coupling network
• Identify amino acids from patterns
• Proline invisible
• Count for consistency
• Sequence assignment requires next step