🧪 1. Using NMR to Determine pKa Values of Single Residues

🎯 Big Idea

NMR can monitor chemical shifts while changing something like:

• pH
• Ligand concentration
• Environmental conditions

This allows you to determine the pKa of individual residues inside a protein.

❓ The Biological Question

The example protein: An acutinase (esterase/lipase)

Lipases typically contain a catalytic triad:

• Histidine
• Aspartate
• Serine

The catalytic mechanism requires histidine to act as a base.

⚠️ Problem:

• Histidine usually has pKa ≈ 6.5
• At pH 5, histidine should be protonated
• If protonated → it cannot function properly in catalysis
• Yet this lipase is still active at pH 5 (even slightly below!)

So how is that possible?

💡 Hypothesis

The pKa of that specific histidine must be shifted inside the protein.

Important principle:

The pKa of a residue in a protein can be very different from its free amino acid value because of its local environment.

🔬 How NMR Solves This

If you can:

1. Identify the NMR signal of the specific residue
2. Measure its chemical shift at different pH values
3. Fit the data to the Henderson–Hasselbalch equation

→ You can extract its pKa value

🧲 Why Histidine is Convenient

Histidine has a very characteristic CH pair chemical shift that:

• Is easy to recognize
• Often does not overlap with other residues
• In this case, there was only one histidine in the protein → perfect system

They used: ¹³C–¹H HSQC spectrum

Axes:

• Carbon chemical shift
• Hydrogen chemical shift

📈 What Happens When You Change pH?

At:

• pH 3.3 → Histidine is protonated → chemical shift changes dramatically
• Higher pH → deprotonated → shift changes again

Both carbon and hydrogen shifts change (in opposite directions), but they follow a titration curve consistent with:

ext{Henderson–Hasselbalch equation}

By fitting the curve:

👉 Measured pKa ≈ 5

🎉 Conclusion

The histidine inside this lipase has:

• A pKa significantly lower than the usual 6.5
• Therefore, at pH 5 it is still mostly deprotonated
• That explains why the enzyme remains catalytically active

🔎 Broader Impact

Special NMR experiments exist to measure pKa for:

• Aspartate
• Glutamate
• Tyrosine
• Others

NMR allows residue-specific thermodynamics inside proteins.

🧬 2. Studying Protein Folding by NMR

Now we move from static properties (pKa) to dynamic processes (folding).

⏱ Problem: Folding is Usually Too Fast

Protein folding timescale:

• Microseconds
• Milliseconds

Even a fast 2D HSQC:

• Minutes (best case)
• Often hours

So real-time folding monitoring is usually impossible.

🐢 Rare Case: Very Slow Folding Protein

Example: Apoplastocyanin

This protein folds over hours.

What was observed?

At time 0:

• Spectrum looks like unfolded protein
• Proton peaks clustered in center
• Side-chain NH₂ signals visible

As time progresses:

• Peaks spread out
• Structured dispersion appears
• After ~2 days → fully folded spectrum

This is a rare but beautiful example of real-time NMR folding observation.

🚀 3. Quenched-Flow NMR (For Faster Folding)

Since most proteins fold too fast, another method is used:

🧪 The Principle: Hydrogen Exchange Protection

Key idea:

Hydrogen bonds protect amide hydrogens from exchanging with solvent.

🔁 The Workflow

Step 1 — Fully unfold protein in D₂O

• All exchangeable hydrogens become deuterium
• No NMR signal from those amides

Step 2 — Trigger folding

• Change buffer conditions
• Allow folding for a short time (ms possible)

Some secondary structures form. Some do not.

Step 3 — Add H₂O

Now:

• Regions already in stable hydrogen bonds → protected → no exchange
• Unfolded regions → exchange → signal disappears

Step 4 — Lower pH

Slow exchange dramatically → “Freeze” the folding state

🧬 What You Get

A folding footprint: Which secondary structure elements existed at that time.

Repeat with different waiting times → You reconstruct the folding timeline.

📚 Beautiful Example: Human Fibroblast Growth Factor

They did the reverse:

• Started in H₂O
• Added D₂O
• Watched signals disappear

Interpretation:

• If a signal disappears → that residue became part of a stable structure

📉 Kinetics per Residue

For each residue:

• Plot signal intensity vs time
• Fit exponential decay
• Extract folding rate

This gives:

Residue-specific folding kinetics

🧱 Folding Order Discovery

In the beta-sheet protein example:

1️⃣ First event:

• N-terminal and C-terminal meet
• Form small β-strand immediately

2️⃣ Second:

• Four β-strands fold

3️⃣ Last:

• Cyan-marked strands fold slowest

They could reconstruct:

• Folding pathway
• Structural hierarchy
• Even generate a “movie” of folding

⚠️ Limitation

If folding occurs:

• Faster than milliseconds

Then:

• Even quenched-flow NMR cannot capture it

Many proteins fold too fast for this method.

🧠 Conceptual Takeaways

1️⃣ Chemical shifts are extremely sensitive

They report on:

• Protonation state
• Hydrogen bonding
• Folding
• Local environment

2️⃣ pKa values in proteins are not intrinsic constants

They depend on:

• Electrostatic environment
• Burial/exposure
• Nearby charges
• Hydrogen bonds

3️⃣ Folding is not uniform

Different structural elements:

• Fold at different speeds
• Can form independently
• Follow specific pathways

4️⃣ NMR is uniquely residue-specific

Unlike many other techniques:

• You see individual amino acids
• You get local thermodynamics
• You get local kinetics

🧩 Overall Summary

This lecture showed two powerful applications of HSQC-based NMR:

1️⃣ Residue-specific pKa determination

• Monitor chemical shift vs pH
• Fit Henderson–Hasselbalch
• Explain altered enzyme activity

2️⃣ Protein folding studies

• Rare real-time monitoring
• Quenched-flow hydrogen exchange
• Residue-specific folding kinetics
• Reconstruction of folding pathways