📘 Protein Chemistry – Day 2 (Part 1)

Focus: Chemical Reactivity of Amino Acids & Post-Translational Modifications (PTMs) (Theory only, based strictly on the lecture transcript )

🔬 Why Do We Care About Side-Chain Chemistry?

The human genome encodes ~30,000 genes, but the number of proteins is far greater due to:

1. Alternative splicing
2. Post-translational modifications (PTMs)

Understanding PTMs requires understanding side-chain chemistry — especially nucleophilicity.

⚛️ Nucleophilicity vs Basicity

• Basicity → Lone pair attacks a proton (H⁺)
• Nucleophilicity → Lone pair attacks something other than hydrogen (e.g., phosphate)

Most PTMs are nucleophilic reactions

🌊 Serine & Threonine

Structure:

Both contain a hydroxyl (-OH) group.

❓ Why are they not very reactive unless the proton is removed?

The OH group has lone pairs, but:

• When protonated → oxygen is neutral
• When deprotonated → oxygen becomes alkoxide (O⁻) → very strong nucleophile

This is why:

• In serine proteases, histidine removes the proton → creates strong nucleophile
• Without deprotonation → very slow reactivity

So you were correct: Serine and threonine become strongly reactive only after proton removal.

🔁 Phosphorylation (Ser, Thr, Tyr)

Adds:

• 2 negative charges
• Extra hydrogen bonding capacity
• Increased steric bulk

Effects:

• Major regulatory PTM
• Alters protein–protein interactions
• Frequently regulates transcription and signaling

🍬 O-Linked Glycosylation (Ser/Thr)

❓ Does O-glycosylation mean glucose attached to oxygen?

Not necessarily glucose specifically. It means:

• A sugar chain is attached to the oxygen atom of Ser/Thr.

The sugar can be various types (mannose, galactose, etc.)

📍 Where does it happen?

• In the ER lumen and Golgi
• Protein must be fully translated
• Occurs after entry into ER lumen

❓ What recognizes the signal sequence?

1. Translation starts in cytoplasm
2. N-terminal signal peptide emerges
3. Recognized by Signal Recognition Particle (SRP)
4. SRP docks to ER membrane
5. Translation continues through ER membrane via translocon

So translation continues — but now into ER lumen.

❓ No consensus sequence — what does that mean?

For O-glycosylation:

• No defined peptide motif around Ser
• Not determined by sequence
• Determined by 3D structure once protein folds

So your idea was right: It depends on structural accessibility, not sequence motif.

🌾 N-Linked Glycosylation (Asn)

Unlike O-linked:

• Has consensus sequence
• Occurs co-translationally (not strictly post-translational!)

Consensus motif:

Asn-X-Ser/Thr

Not all sites are modified — but modification requires this motif.

🔗 Transglutaminase Crosslinks

Glutamine can form covalent crosslinks with:

• Lysine
• Cysteine

Enzyme involved:

Transglutaminase

Used industrially (e.g., plant-based meat structure).

⚡ Lysine

Primary amine (NH₃⁺)

• Good nucleophile when deprotonated
• Reactivity increases at high pH

PTMs:

• Acetylation
• Methylation
• Ubiquitination
• Biotinylation
• Lipoylation

❓ What enzyme does biotinylation?

Biotin is attached enzymatically to lysine residues by biotin ligases (biotin protein ligase). It is a PTM

🧬 Histone Modifications

Lysine & arginine methylation/acetylation:

• Neutralizes positive charge
• Weakens DNA binding
• Regulates gene expression

🧲 Histidine

Contains imidazole ring

At physiological pH:

• One nitrogen protonated
• One has lone pair

❓ How does His-tag work?

Not active site chemistry.

His-tag:

• Several histidines engineered at N- or C-terminus
• Lone pair electrons coordinate to Ni²⁺
• Forms coordination bond
• Lowering pH protonates histidine → releases from nickel

It does NOT donate proton to serine in this context. That happens in enzyme active sites — unrelated to His-tag purification.

🔪 Proteolytic Cleavage

Yes — occurs after protein synthesis

It is considered a post-translational modification

Example: signal peptide removal, pro-protein activation.

🧪 Methionine Removal

Yes — removal of N-terminal Met is:

• A post-translational modification
• Very common

🔥 Cysteine — The Most Reactive Amino Acid

Contains SH group.

❓ Why is cysteine a strong nucleophile at physiological pH?

pKa ≈ 8–8.3

Physiological pH ≈ 7.4

Using Henderson-Hasselbalch:

When pH is 1 unit below pKa:

→ ~10% is deprotonated

So:

• ~10% exists as thiolate (S⁻)
• Thiolate is extremely strong nucleophile
• Therefore high reactivity even at pH 7.4

Your understanding was correct: Yes, roughly ~10% negatively charged explains strong nucleophilicity.

🔗 Disulfide Bonds

Oxidation:

2 Cys → Cys-S-S-Cys

Reduction:

• DTT
• β-mercaptoethanol
• TCEP

🟡 Ellman’s Reagent

Used to detect free cysteine

Reacts with free thiols → yellow product

Absorbance max: 412 nm

❓ Does yellow mean non-covalently bound?

Small correction:

Yellow means free thiol (not involved in disulfide bond)

It does NOT mean “non-covalently bound” — it means:

The cysteine is not covalently linked via disulfide bridge.

🧠 Quick Concept Summary

🎯 Final Clarifications

You misunderstood only two minor points:

1. His-tag ≠ active site chemistry
2. Ellman’s reagent detects free thiols, not “non-covalent binding”

Everything else you interpreted correctly.