🧲 1. Why is Protein NMR Different & Difficult? (Pages 1–5)

🔹 Problem I: Signal Overlap

Page 2 – Single Amino Acid

A single amino acid → easy spectrum. Each proton (HN, Hα, Hβ, etc.) gives a separate peak.

No ambiguity. No overlap.

Page 3 – Hexapeptide

Now peaks overlap heavily.

Why?

• Many similar chemical environments
• Many NHs around 8 ppm
• Many aliphatic protons 0–4 ppm
• Aromatics pile up 6–8 ppm

Result: impossible to interpret in 1D.

Page 4 – 148 aa Calmodulin

Massive 1D overlap.

Solution: Spread information into more dimensions. Instead of 1D → use 2D and 3D.

Think of it like:

• 1D = traffic jam
• 2D = spread cars onto two roads
• 3D = multi-level parking

Page 5 – Size Dependence

HSQC spectra of proteins from 42 aa to 377 aa show:

• More residues → more peaks
• Larger proteins → broader peaks (relaxation)
• Eventually impossible without isotope labeling

🧲 2. Problem II: T₂ Relaxation (Pages 6–9)

Page 6 – T₂ Relaxation

Large proteins tumble slowly.

Slow tumbling → short T₂ → broad peaks → low intensity.

Key concept:

• Small molecule → sharp peaks
• Large protein → broad weak peaks

Page 7 – Deuteration

Replace many ¹H with ²H (deuterium).

Why it helps:

• Fewer dipolar interactions
• Slower relaxation
• Narrower peaks

Comparison shown:

• Fully protonated → messy
• 75% deuterated → clean

Page 8 – TROSY

TROSY selects the slowest relaxing component of the multiplet.

Important idea:

• In large proteins, different relaxation pathways partially cancel.
• TROSY isolates the best one.

Result:

• Much sharper peaks
• Works well > 30 kDa

Page 9 – TROSY + Deuteration

Together → powerful. Even 45 kDa protein becomes feasible.

🧬 3. Disordered Proteins (Page 10)

α-synuclein vs calmodulin.

Disordered proteins:

• Narrow chemical shift dispersion
• All HN clustered around ~8 ppm

Very heavy overlap.

🧪 4. Sample Requirements (Page 12)

Critical:

• 1 mmol material

• 0.2 mM concentration

• Stable for weeks
• Isotope labeling depends on size:

  • 50 aa → ¹⁵N

  • 100 aa → ¹⁵N/¹³C

  • 300 aa → ²H/¹⁵N/¹³C

• Protein must be folded & monodisperse

Without good sample → no NMR.

🧠 5. Assignment Strategy Overview (Page 13)

Workflow:

1. Sample preparation
2. Resonance assignment (huge time)
3. NOE collection
4. Structure calculation
5. Functional/dynamics studies

Assignment is foundation.

🧭 6. Homonuclear Assignment – Small Peptides (Pages 15–30)

Used when no isotope labeling.

Required spectra (Page 16):

• TOCSY (80–90 ms)
• NOESY (50–60 ms)
• 2QF-COSY

🧬 6A. Understanding COSY vs TOCSY (Pages 17–19)

🔹 COSY

Shows direct J-couplings only (usually 2–3 bonds).

So: Hα ↔ Hβ Hβ ↔ Hγ

But NOT: Hα ↔ Hγ (unless directly coupled)

COSY = nearest neighbors in bond network.

🔹 TOCSY

Shows entire spin system via relayed magnetization.

All protons within same residue connect.

Example: HN → Hα → Hβ → Hγ → Hδ → etc.

TOCSY = entire amino acid fingerprint.

🧬 6B. What is a Spin System? (Page 17)

Important: No J-coupling across peptide bond.

Each amino acid = independent spin system.

This is the key assumption for assignment.

🧬 6C. Sequential Assignment Procedure (Pages 20–28)

Step 1 – Number HN/Hα peaks (Page 20–22)

Arbitrarily label them.

Step 2 – Use TOCSY

Find all peaks belonging to same spin system.

Step 3 – Identify residue type (Page 23)

Examples:

• Gly: 2 Hα, nothing else
• Ala: Hα + CH₃
• Thr: 2 Hα-like + CH₃
• Val: Hα + Hβ + 2 CH₃
• AMX pattern: Ser, Cys, Asp, Asn, His, Phe, Tyr, Trp
• Lys/Arg: many signals 3–3.5 ppm
• Pro: no HN → breaks walk

This is pattern recognition.

🧬 6D. Sequential NOE Walk (Page 26)

Critical rule:

HN(i) shows NOE to:

• Hα(i-1)
• Hβ(i-1)

NEVER forward.

So you connect: Residue i ↔ residue i-1

This is the “sequential walk”.

Important note:

HN-HN NOEs appear only in α-helices.

In β-sheets → interstrand NOEs appear (Page 31).

These can confuse assignment.

⚠️ Pitfalls (Page 30)

• Overlap
• Prolines break walk
• Secondary structure gives extra NOEs
• Very difficult without labeling

🧪 7. ¹⁵N Labeling Only (Pages 32–33)

Use ¹H–¹⁵N HSQC.

Each amide = one peak.

Better dispersion because ¹⁵N axis spreads signals.

🔹 3D ¹⁵N-edited TOCSY (Page 33)

Dimensions:

1. HN
2. ¹⁵N
3. side-chain proton

Interpretation: Take one HSQC peak → look into 3rd dimension → see full spin system.

This removes overlap in HN dimension.

Limitation: Weak for larger proteins.

🧪 8. Triple Resonance Experiments (Pages 34–41)

This is the modern method.

Requires ¹⁵N/¹³C labeling.

🔹 HNCA vs HN(CO)CA (Pages 35–39)

HNCA detects:

Cα(i) AND Cα(i-1)

HN(CO)CA detects:

ONLY Cα(i-1)

So:

If peak appears in both → Cα(i-1) If only in HNCA → Cα(i)

This is the backbone linking logic.

🔹 HNCO / HN(CA)CO

Same idea but for carbonyl (C').

🔹 CBCA(CO)NH / CBCANH

Same logic for Cα and Cβ.

Gives amino acid identity.

🧲 9. How to Interpret 3D Spectra (Page 37 – Strips)

A 3D spectrum has 3 axes:

Example:

• ¹H
• ¹⁵N
• ¹³C

Visualization method: You slice at one HSQC peak → generate a “strip”.

Each strip: = one residue

Then compare neighboring strips: Find matching Cα(i-1) to Cα(i)

That builds sequence.

Strip comparison is like matching puzzle pieces.

🧪 10. Chemical Shift Patterns (Page 42)

Cα vs Cβ shifts cluster by residue type.

Example:

• Gly: low Cα
• Thr: high Cβ
• Val/Ile: characteristic pattern

This helps residue identification.

🧬 11. Side Chain Assignment (Pages 43–45)

Experiments:

• H(C)CH-TOCSY
• (H)CCH-TOCSY
• H(CCCO)NH
• HCCH-type

These transfer magnetization across carbon network.

Used for:

• Aliphatic side chains
• Aromatics (very difficult)

Assignment often takes 50% of project time.

🧾 12. Complete Experiment Set (Page 46)

Typical backbone set:

• HNCA
• HN(CO)CA
• HNCO
• HN(CA)CO
• CBCA(CO)NH
• CBCANH
• HBHA(CBCACO)NH
• HCCH-TOCSY

Exact choice depends on protein size.

📚 13. Literature Assignments (Page 47)

BioMagResBank (BMRB) http://www.bmrb.wisc.edu

Contains deposited assignments.

🎯 Big Picture Summary

Protein NMR challenges:

1. Signal overlap
2. T₂ relaxation
3. Large number of resonances

Solutions:

• 2D/3D experiments
• Isotope labeling
• Deuteration
• TROSY
• Triple resonance backbone linking

Assignment logic:

Spin system → sequential connection → backbone linking → side chain completion → NOEs → structure.