🧬 From Spin Systems to Sequence Assignment

In Part 1, you identified individual spin systems (e.g., alanine, leucine, AMX, alanine). But identifying spin systems is not enough.

You still need to answer:

• Which residue number is this?
• In what order do they appear in the sequence?
• Which alanine is which?

This is where the sequential walk comes in.

🔗 The Key Insight: NH Knows Its Neighbor

Nature helps us.

An amide proton (HN) is:

• Close in space to its own Hα
• Also close in space to the Hα of the previous residue
• ❌ Not close to the next residue’s Hα (distance too long)

Why does this matter?

Because NOESY detects through-space interactions (dipolar coupling). So in a NOESY spectrum, you usually see:

• HN(i) ↔ Hα(i) (intra-residue)
• HN(i) ↔ Hα(i−1) (sequential)
• Often HN(i) ↔ Hβ(i−1)
• ❌ Usually not HN(i) ↔ Hα(i+1)

That directionality gives you sequence information.

🧪 TOCSY vs NOESY in This Context

The file illustrates:

• Upper triangle → TOCSY
• Lower triangle → NOESY (This combined spectrum doesn’t physically exist — it’s pedagogical.)

What’s the difference here?

• TOCSY → shows atoms connected through bonds (within spin system)
• NOESY → shows atoms close in space (between residues)

TOCSY identifies spin systems. NOESY connects them.

🚶 The Sequential Walk

This is the core concept.

Step 1:

Pick an HN peak.

Step 2:

Look in NOESY:

• Which Hα does it correlate with?

You’ll find:

• One Hα from the same residue
• One Hα from the previous residue

Step 3:

Go to that previous residue’s spin system.

Step 4:

Repeat.

You “walk” residue by residue through sequential NOEs.

In the example:

Spin system order discovered:

1 → 4 → 3 → 2

This corresponds to a tetrapeptide fragment:

Ile/Leu – Ala – AMX – Ala

🔍 Matching to the Protein Sequence

Once you know the order of amino acid types:

1. Search the protein sequence.
2. Find where this fragment occurs uniquely.

Example from file:

Found:

Ala – AMX – Ala – Ile

It appears only once.

If that alanine is residue 17, then:

Boom. 🎯 Four residues assigned.

Repeat until the protein is assigned.

⚠️ Pitfalls in Sequential Assignment

Very important section.

1️⃣ Signal Overlap

If two Hα shifts are identical:

• Their NOEs overlap
• You cannot distinguish them

2️⃣ Proline

Proline has:

• No amide proton (HN)

So the sequential walk stops at Proline.

3️⃣ Secondary Structure Creates Extra NOEs

This is a BIG source of confusion.

🌀 Alpha Helix NOE Pattern

In α-helix:

You see:

• HN(i) ↔ Hα(i−1) (sequential)
• HN(i) ↔ HN(i−1)
• HN(i) ↔ Hα(i−3)
• Sometimes HN(i) ↔ Hα(i−4)

Important: The i → i−3 distance is nearly as short as the sequential one.

So the NOE intensity may be similar.

These long-range NOEs are:

• Excellent for structure determination
• Confusing for assignment

🧵 Beta Sheet NOE Pattern

In β-sheets:

You see:

• Sequential HN(i) ↔ Hα(i−1)
• ❌ No i−3 (too far)

BUT:

• HN can show NOEs to residues across the strand

These are inter-strand NOEs.

Sequential NOEs are usually stronger (shorter distance), but intensity alone cannot always be trusted.

🧪 When Homonuclear Spectra Are Not Enough

For intermediate-size proteins:

• Fully homonuclear assignment may fail
• But full 13C/15N labeling may not be necessary

Solution:

15N labeling only

Why?

Nitrogen chemical shifts are well dispersed.

Effect:

• Overlapping HN peaks can now be separated
• But aliphatic hydrogens (Hα, Hβ) are still overlapped

📊 3D 15N-Edited TOCSY / NOESY

Now spectra are 3D.

Dimensions:

• H (direct)
• H (indirect)
• 15N (third dimension)

What happens?

Each HN peak is “lifted” into the 15N dimension.

If two HNs overlap in 2D, they likely have different 15N shifts.

So in 3D:

• Their spin systems separate.

Huge advantage.

📦 How Do You View a 3D Spectrum?

You don’t stare at a cube.

You extract:

Strips

One strip per residue.

Software identifies:

• HN frequency
• Displays cross peaks in a slice

So instead of chaos, you see organized strips.

Very powerful for assignment.

🧠 Key Concepts to Remember

Sequential NOE

HN(i) ↔ Hα(i−1)

Sequential walk

Following NOEs residue by residue.

Assignment strategy

1. Identify spin systems (TOCSY)
2. Connect via NOESY
3. Match fragment to sequence

Pitfalls

• Overlap
• Proline
• Helical i−3 NOEs
• β-sheet cross-strand NOEs

3D rescue

15N-edited experiments separate overlapping amides.

🎯 Big Picture

This entire method connects:

Spin System Identification → Sequential NOEs → Fragment Identification → Matching to Sequence → Residue-Specific Assignment

Only once this is done can full 3D structure determination proceed.