🎥 Lecture 5 – Video 1

What Else Can We Do with Protein NMR?

This lecture is the big-picture introduction to the final part of the protein NMR course: ➡️ Studying protein function once we know (or don’t even know!) the structure.

It sets the stage for everything that follows in the series and explains how NMR moves from “What does it look like?” to “What does it do?”

🧬 From Structure to Function

Earlier in protein NMR, the focus was structure determination. Now the focus shifts to:

🔍 How proteins work in solution

Important point:

• Studying protein function usually requires resonance assignment
• It does not always require a full 3D structure

So assignment = often essential Structure = helpful, but not always mandatory

🔗 1. Ligand Binding – The Core of Protein Function

One of the most important aspects of protein function is:

How proteins interact with other molecules

What are ligands?

Ligands can be:

• 🧪 Small molecules (substrates, inhibitors)
• 🧬 Other proteins
• 🧫 Membranes
• 📡 Signaling partners

Much of what proteins do in cells is binding.

🧭 How NMR Studies Ligand Binding

There are multiple strategies, depending on the strength and nature of interaction.

🧱 A. Stable Complexes → Structure Determination

If the protein–ligand complex is stable:

• You can measure NOEs between protein and ligand
• These NOEs provide distance restraints
• You can calculate the structure of the complex

This is the most detailed level of information: You see exactly how the ligand sits in the binding pocket.

📍 B. Chemical Shift Perturbations (CSPs)

Even if the complex is not extremely stable:

• Ligand binding causes chemical shift changes
• You observe this in the ¹⁵N-HSQC
• You get a “footprint” of the ligand on the protein surface

This tells you:

• Which residues are affected
• Where the binding site likely is

Even without structure calculation, this gives powerful spatial information.

💧 C. Hydrogen Exchange Changes

Ligand binding can:

• Protect residues from solvent
• Change local stability
• Alter exchange rates

By monitoring hydrogen exchange, you can detect:

• Stabilization
• Structural rearrangements

🧲 D. PREs (Paramagnetic Relaxation Enhancements)

If you introduce a paramagnetic label:

• Nearby nuclei experience enhanced relaxation
• Signals broaden or disappear

This gives long-range distance information beyond NOE limits

🎯 E. Focusing on the Ligand Instead

Sometimes the ligand is more interesting than the protein.

Then you can use:

• Saturation transfer experiments
• Transferred NOEs (mentioned but not covered in this series)

This is especially useful when:

• Protein is large
• Ligand is small
• Complex is transient

🔄 2. Protein Dynamics

Proteins are not static objects.

Key questions:

• Which parts are flexible?
• Which parts are rigid?
• Does flexibility change upon binding?
• Does environment alter dynamics?

Part five of the lecture series focuses on studying molecular dynamics by NMR

NMR is uniquely powerful here because it:

• Observes proteins in solution
• Measures motions across multiple timescales
• Detects subtle conformational fluctuations

🧩 3. Protein Folding

Protein folding is described as:

An intriguing problem

Challenge:

• Folding is often too fast for classical NMR

But:

• There are ways to study aspects of folding
• Later parts (especially part seven) address what is possible

NMR can probe:

• Stability
• Folding intermediates
• Protection patterns

💦 4. Hydrogen Exchange

Hydrogen exchange is highlighted as:

An important tool for studying protein behavior in solution

What does it measure?

• Exchange of backbone amide hydrogens with solvent
• Sensitivity to:
  • Hydrogen bonding
  • Structural protection
  • Stability
  • Ligand binding

Used in:

• Functional studies
• Folding studies
• Stability mapping

Covered especially in:

• Part 2B
• Part 7

⚡ 5. Measuring pKa Values of Individual Side Chains

A very powerful capability of NMR:

Determining pKa values of single residues inside proteins

Why is this important?

Because:

• pKa shifts reveal local electrostatic environment
• Catalytic residues often have perturbed pKa values
• Protonation states affect function

NMR can track chemical shift changes as pH varies and:

• Extract titration curves
• Determine residue-specific pKa values

🛠 The Workhorse: ¹⁵N-HSQC

This is described as the:

Power tool of protein NMR

Why is it so powerful?

The ¹⁵N-HSQC:

• Detects every backbone NH group
• Gives one cross peak per residue (mostly)
• Acts like an antenna on every amino acid

So:

Each peak = one specific residue If you have assignment → you know exactly which one.

It becomes:

• A fingerprint of the protein
• A highly sensitive reporter of structural change
• The central spectrum for most functional studies

Almost every experiment in this series builds on this.

🧠 Big Conceptual Takeaway

Protein NMR is not just about structure.

It allows you to probe:

• 🔗 Binding
• 🔄 Dynamics
• 🧩 Folding
• 💧 Hydrogen exchange
• ⚡ pKa values
• 🧲 Long-range interactions
• 🎯 Ligand-focused experiments

And most of this can be done:

• In solution
• Under near-physiological conditions
• Often without needing a full structure

🧭 Perspective of the Series

The lecture serves as a roadmap:

🌟 Final Conceptual Insight

Think of protein NMR as layered information:

1️⃣ Structure → where atoms are 2️⃣ Binding → what interacts 3️⃣ Dynamics → how it moves 4️⃣ Folding → how it forms 5️⃣ Protonation → how chemistry shifts

It’s not one technique. It’s a toolbox.

And the ¹⁵N-HSQC is the control panel that connects everything.