Protein NMR Beyond Structure – Reinhard Wimmer

This lecture explains how NMR is used not just to determine structure — but to study function, dynamics, binding, folding, and energetics.

🧭 OVERVIEW

The lecture is divided into:

• Part I – Introduction
• Part IIA – Ligand binding by chemical shift perturbation
• Part IIB – Hydrogen/Deuterium exchange
• Part III – Ligand binding from NOEs
• Part IV – Ligand binding from the ligand perspective (STD-NMR)
• Part V – Protein dynamics
• Part VI – Paramagnetic relaxation enhancement (PRE)
• Part VII – pKa values & protein folding

🟢 PART I – INTRODUCTION

📄 Page 1 – Title

Introduction to using Protein NMR beyond structure determination.

📄 Page 2 – The Workflow of NMR Studies

The classical structure determination path:

1. Sample preparation
2. Resonance assignment
3. NOEs & couplings
4. Structure calculation

➡️ This consumes huge NMR time and computer time.

But once structure is known → NMR can study:

• Function
• Dynamics
• Mechanism

📄 Page 3 – What Can NMR Study?

Beyond structure:

• Ligand binding
• Molecular dynamics
• Protein folding
• pKa values
• Hydrogen exchange

Each topic corresponds to later lecture parts.

📄 Page 4 – Protein–Ligand Interactions

Methods listed:

• Protein–ligand NOEs (stable complexes only)
• Chemical shift changes
• Hydrogen exchange changes
• PREs
• Saturation transfer (ligand-focused)

Important concept: Different NMR observables report on different aspects of binding.

📄 Page 5 – 15N-HSQC: The “Power Tool” 🔥

The HSQC spectrum:

• X-axis: 1H
• Y-axis: 15N
• One peak per backbone NH (mostly)

This is a fingerprint of the protein.

The image shows:

• Structure of a protein
• Corresponding HSQC

Key idea: If something changes in structure or environment → peaks move.

📄 Page 6 – Take-Home Messages

NMR can study:

• Binding thermodynamics
• Structural aspects
• Molecular motion
• Folding
• pKa

HSQC = extremely sensitive probe of environment changes.

🟡 PART IIA – CHEMICAL SHIFT PERTURBATION (CSP)

📄 Page 7 – Title

📄 Page 8 – Concept of CSP

Chemical shift reflects:

• Time-averaged local electronic environment

When ligand binds: → Local environment changes → Chemical shifts change

So: Map shifted residues → identify binding site.

📄 Page 9 – Binding Thermodynamics Refresher

Reaction: P + L ⇌ PL

Parameters:

• kon (binding rate)
• koff (dissociation rate)
• Ka = kon/koff
• Kd = 1/Ka

Diffusion-limited kon ≈ 10⁷ M⁻¹s⁻¹.

If conformational change required → slower.

📄 Page 10 – Experimental Setup

Add ligand → record HSQC.

Outcomes:

• No change → no binding
• Peak shifts → binding
• Map shifts → binding site
• Fit shifts → estimate Kd

📄 Pages 11–12 – Chemical Exchange Regimes

Important NMR concept.

Exchange rate (kex) compared to chemical shift difference (Δν):

1️⃣ Slow exchange (kex << Δν)

• Two peaks visible

2️⃣ Fast exchange (kex >> Δν)

• One peak moving

3️⃣ Intermediate exchange

• Peak broadening / disappearance

📄 Pages 13–14 – Visual Examples

Page 13: Slow exchange spectra at different ligand ratios: Separate free and bound peaks.

Page 14: Fast exchange titration: Peaks gradually shift position.

📄 Page 15 – Ligand Titration Curves

Strong binding:

• Steep curve
• Saturates quickly

Weak binding:

• Gradual curve

Important: CSP works best for Kd > 0.1 mM.

📄 Pages 16–17 – Calculating Ka

In fast exchange:

Δδobs / Δδbound = fraction bound

Using binding equations for 1:1 complex: You can solve for Ka.

Key insight: Chemical shifts can quantify thermodynamics.

📄 Pages 18–23 – Case Study: Plectasin

Study: How does antimicrobial peptide plectasin bind lipid II?

Steps:

1. Add DPC micelles (membrane mimic)
2. Observe binding
3. Add lipid II

Findings:

• One hydrophobic end inserts into micelle
• Lipid II binds in semi-ring around protein
• Chemical shift mapping shows binding surface

ΔG = −27 kJ/mol.

📄 Page 24 – CSP Pros & Cons

Advantages:

• Simple
• No complex theory
• Gives Ka
• Works for all exchange regimes

Disadvantages:

• No structural detail
• Secondary effects cause shifts
• Not a structure of complex

Rule: Binding site + neighboring residues always shift.

🟠 PART IIB – H/D EXCHANGE

📄 Pages 25–26 – Concept

Amide hydrogens exchange with solvent.

In D2O: NH → ND Peak disappears in HSQC.

Exchange rate depends strongly on pH.

📄 Page 27 – HSQC Visibility

Visible: NH Invisible: ND

Thus: Loss of peak intensity reports exchange.

📄 Page 28 – Experimental Setup

1. Freeze-dry protein
2. Dissolve in D2O
3. Record HSQC over time
4. Monitor intensity decay

📄 Page 29 – Protection Factor

Hydrogen bonds protect NH from exchange.

Protection factor = observed / expected rate.

High protection indicates:

• Secondary structure
• Burial
• Ligand binding

📄 Pages 30–31 – Binding Surface Example

CBP21 + chitin

Observation: Certain residues protected upon substrate binding.

Mapped onto structure: Defines substrate binding surface.

📄 Page 32 – Take-Home Messages

H/D exchange useful for:

• Detecting secondary structure
• Detecting ligand epitopes
• Studying folding

🔴 PART III – NOEs FOR LIGAND BINDING

📄 Pages 33–34

NOEs between protein and ligand: → Provide distance restraints → Can determine structure of complex

Requires: Stable complex (high Ka, slow dissociation).

📄 Pages 35–36 – Example

Fatty acid binding protein: NOEs define precise orientation of fatty acid.

📄 Page 37 – Take-Home

NOEs = structural information But only if binding strong enough.

🟣 PART IV – STD-NMR (Ligand Perspective)

📄 Pages 38–40 – Saturation Transfer

Saturate protein resonance. Saturation spreads via spin diffusion. Transfers to ligand in contact.

Ligand dissociates: Carries saturation with it.

📄 Pages 41–43 – Example: NA₂ & RCA120

STD spectrum shows: Strong signals = ligand atoms closest to protein.

Weak/no signal = far from protein.

Thus: Maps ligand binding epitope.

📄 Page 44 – STD Summary

Advantages:

• No protein assignment needed
• Works at low purity
• No isotope labeling required

Limitation: Gives no protein information.

🔵 PART V – PROTEIN DYNAMICS

📄 Pages 45–47 – Timescales

Protein motions:

10⁻¹² s – side chain rotation 10⁻¹⁰–10⁻¹¹ s – loop motions 10⁻⁹–10⁻⁸ s – overall tumbling 10⁻⁷–10⁻³ s – slow breathing

📄 Page 48 – Model-Free Approach

Parameters:

• Overall tumbling (τm)
• Order parameter S² (0–1)

S² = 1 → rigid S² = 0 → fully flexible

📄 Page 49 – T1 & T2

T1, T2 relaxation: Depend on mobility.

T1/T2 ratio estimates tumbling.

📄 Pages 50–54 – Case Study: Calmodulin

Mutation F141L:

• Increases flexibility in C-lobe
• Linker region extended

Measured using: 15N{1H}-NOE.

📄 Page 55 – Take-Home

Relaxation measures:

• Overall motion
• Internal flexibility

NOE distinguishes rigid vs flexible regions.

🟤 PART VI – PRE

📄 Pages 56–61 – Basics

Paramagnetic centers (unpaired electrons) → Strong magnetic moment → Enhance relaxation → Signal attenuation

PRE ∝ r⁻⁶ (distance dependent)

📄 Pages 62–67 – Theory

PRE arises from:

• Dipole–dipole interactions
• Electron–nucleus interactions

Requires:

• Paramagnetic label (Gd³⁺, Mn²⁺, nitroxide)
• Diamagnetic control

📄 Pages 68–72 – Example: Anoplin

Measured PRE distances in micelle. Determined insertion depth.

PDB 2MJQ.

📄 Pages 73–77 – Transient Complexes

PRE detects: Very low-populated states (~0.5%).

Example: Multiple transient geometries explain PRE data.

📄 Pages 78–79 – PRE Summary

PRE useful for:

• Long-range constraints
• Solvent accessibility
• Transient states

Analogy: Like fluorescence quenching.

🟡 PART VII – pKa & Folding

📄 Pages 80–82 – pKa by NMR

Monitor chemical shifts vs pH.

Protonation changes shift. Fit curve → get pKa of individual residues.

Example: Active-site His in cutinase.

📄 Pages 83–84 – Real-Time Folding

Most proteins fold too fast for NMR.

Rare slow folder: Apoplastocyanin.

Observed folding over hours.

📄 Pages 85–86 – Quenched-Flow NMR

Strategy: Allow folding for defined time. Trigger H/D exchange. Freeze state. Measure protection.

Snapshots of intermediates.

📄 Pages 87–89 – Folding Example

Human fibroblast growth factor.

Observation: As folding time increases: More H-bonds form. Less exchange. HSQC intensity decreases.

🎯 FINAL TAKE-HOME

Protein NMR can study:

✅ Ligand binding (CSP, NOE, STD) ✅ Binding thermodynamics ✅ Binding epitopes ✅ Protein dynamics (T1, T2, NOE) ✅ Paramagnetic long-range effects ✅ pKa values ✅ Folding intermediates

HSQC remains the central tool throughout.