Lecture 2 Paper 3

Protein chemistry

🧬 Protein Posttranslational Modifications (PTMs): The Chemistry

C. T. Walsh, S. Garneau-Tsodikova, G. J. Gatto Jr.

1️⃣ Introduction – Why PTMs Matter 🧠

The proteome is vastly larger than the genome because proteins are chemically modified after translation. Roughly 5% of eukaryotic genes encode PTM enzymes, including:

~500 kinases
~150 phosphatases
~500 proteases

Two fundamental PTM categories (Scheme 1):

Covalent addition of a chemical group to an amino-acid side chain
Covalent cleavage/rearrangement of the peptide backbone

PTMs:

Expand chemical functionality
Control activity, localization, stability
Enable signal integration and epigenetic regulation

2️⃣ Covalent Addition: The Main Acts ⚗️

Five dominant PTM chemistries account for most cellular regulation.

🔹 2.1 Phosphorylation (Ser, Thr, Tyr, Asp, His)

ATP-dependent transfer of phosphate to nucleophilic –OH or –N groups
Central to signal transduction
Reversible via phosphatases

Key points:

Kinases often activated by autophosphorylation
MAP kinases require dual phosphorylation
One kinase (e.g., PKA) can modify >100 substrates

💡 Prototype reversible PTM

🔹 2.2 Acylation (Lys, N-termini, Cys)

Major forms:

Acetylation (acetyl-CoA → Lys ε-NH₂)
Ubiquitylation (Ub attached via isopeptide bond)
SUMOylation
Biotinylation, lipoylation
S-palmitoylation (thioester, reversible)

Functional roles:

Histone acetylation → chromatin opening
Ubiquitylation → proteasomal degradation
Lipidation → membrane targeting

⚖️ Competition example: Lys residues on p53 can be either acetylated or ubiquitylated, determining protein lifetime

🔹 2.3 Glycosylation 🍬

N-linked glycosylation:

Asn-X-Ser/Thr motif
Occurs in ER during protein translocation

O-linked glycosylation:

Ser/Thr
Often regulatory (e.g., Notch signaling)

Key insight:

Incomplete occupancy → massive glycoform diversity (Prion protein ≈ 52 glycoforms)

🔹 2.4 Thiol–Disulfide Chemistry (Cys)

Disulfide bonds stabilize extracellular proteins
Redox-regulated by thioredoxin/glutathione systems
Includes S-nitrosylation (NO signaling)

🔹 2.5 Alkylation (Methylation & Prenylation)

Methylation (SAM-dependent):

Lys and Arg (histones!)
Mono-, di-, trimethylation = distinct signals

Prenylation:

Farnesyl (C15) or geranylgeranyl (C20)
Targets Ras/Rho/Rab GTPases to membranes

📌 CaaX rule:

X = Ser/Ala → farnesyl
X = Leu → geranylgeranyl

3️⃣ Covalent Addition: The Supporting Cast 🧪

Less common but chemically fascinating PTMs.

🔹 Protein Hydroxylation

Fe²⁺/O₂-dependent monooxygenases
Examples:
- 4-Hydroxyproline → collagen stability
- 5-Hydroxylysine → further glycosylation
- Pro/Asn hydroxylation → oxygen sensing (HIF-1α)

🔹 Cross-Linking

Transglutaminases
Gln–Lys isopeptide bonds
Structural stabilization (e.g., fibrin)

🔹 Oxidative Cys Chemistry

Sulfenic acids
Redox signaling intermediates
Can be reversible or lead to damage

4️⃣ Cataloguing PTMs 📊

PTMs can be classified by:

Residue modified (15 of 20 amino acids)
Cosubstrate used (ATP, SAM, acetyl-CoA, NAD⁺…)
Functional outcome

Table 1 in the paper provides a master list of known side-chain modifications .

5️⃣ Multiple & Tandem PTMs 🔗

Proteins often carry many PTMs simultaneously.

Examples:

Abl kinase: 11 phosphorylation sites → >40 million theoretical isoforms
Histone tails: acetylation, methylation, phosphorylation, ubiquitylation

🧬 Histone code: PTMs are written, read, and erased in defined patterns to regulate transcription

Threshold effects:

≥4 ubiquitins required for proteasome targeting

6️⃣ Reversible vs Irreversible PTMs 🔄

Reversible:

Phosphorylation
Acylation
Glycosylation
Disulfides

Irreversible:

Prenylation (thioether)
Glu → Gla carboxylation (vitamin K-dependent)
Pro/Asn hydroxylation
Proteolysis

🧠 Design principle: Reversibility reflects signaling vs structural commitment

7️⃣ Controlled Proteolysis ✂️

Proteasome degradation via polyubiquitin
Temporal control (e.g., cyclins in cell cycle)
E1–E2–E3 enzyme cascade

PTMs regulate both substrate and ligase activity

8️⃣ Autocleavage & Protein Splicing 🧩

Intein-mediated splicing:

Internal peptide excised
Exteins ligated via thioester intermediates
Applications in protein engineering

9️⃣ Peptide Bond Rearrangement without Cleavage 🌈

🔹 GFP Chromophore Formation

Internal Ser-Tyr-Gly tripeptide cyclizes
Dehydration + oxidation → fluorescent chromophore
Requires native protein fold

Variants:

GFP → green
DsRed → red via extended conjugation

🔹 Methylene-Imidazolone (MIO) Cofactor

Formed from Ala-Ser-Gly tripeptide
Creates electrophilic cofactor
Enables ammonia-lyase chemistry (His/Phe deaminases)

🔟 Conclusions 🎯

200 PTMs known, expanding protein chemistry far beyond genetics
PTMs:
- Enable new catalytic functions
- Control signaling, localization, stability
- Provide epigenetic adaptability
The proteome is a dynamic chemical system, not a static gene product

🧠 Final Exam Take-Home Messages

PTMs are chemically rational, not random
Reversibility ≈ signaling
Irreversibility ≈ commitment
Combinatorial PTMs multiply functional diversity
Protein chemistry is a post-genetic logic layer

Quiz

Score: 0/29 (0%)

Q0. What is the primary reason the proteome is far more diverse than the genome?

Alternative RNA splicing alone

Posttranslational covalent modification of proteins

Protein misfolding during translation

Random chemical damage to proteins

Q1. Which two broad chemical categories encompass all posttranslational protein modifications described by Walsh?

Oxidation and reduction

Addition of cofactors and removal of cofactors

Covalent side-chain addition and peptide backbone cleavage/rearrangement

Enzymatic and non-enzymatic reactions

Q2. Which amino acid side chains are most commonly phosphorylated in eukaryotic signaling?

Ser, Thr, and Tyr

Asp and Glu

Lys and Arg

Cys and His

Q3. Why is phosphorylation considered the prototype of reversible posttranslational modification?

Because it does not change protein charge

Because it is chemically unstable

Because it is reversed by dedicated phosphatases

Because it only occurs once per protein

Q4. What chemical feature distinguishes ubiquitylation from most other acylations?

It forms a glycosidic bond

It uses NAD+ as a cosubstrate

It forms an isopeptide bond resistant to proteases

It modifies only N-terminal residues

Q5. Which statement best explains the enormous heterogeneity of glycoproteins?

Glycosylation is genetically encoded

Each glycosylation step proceeds with incomplete efficiency

Sugars spontaneously rearrange after attachment

Glycosylation occurs only in the cytosol

Q6. What is the functional consequence of prenylation of small GTPases such as Ras or Rho?

Increased protein solubility

Targeting to membrane surfaces

Direct activation of catalytic activity

Protection from oxidation

Q7. Why is S-palmitoylation considered reversible whereas prenylation is not?

Palmitoylation uses ATP

Palmitoylation forms a thioester bond

Prenylation occurs in the nucleus

Prenylation modifies backbone atoms

Q8. Which PTM is central to the concept of the histone code?

Hydroxylation

Phosphopantetheinylation

Acetylation and methylation of Lys and Arg

Disulfide bond formation

Q9. Why does Walsh emphasize combinatorial PTMs as a systems-level challenge?

They are chemically unstable

They generate factorial numbers of possible protein isoforms

They occur only in bacteria

They prevent protein degradation

Q10. What is the biochemical threshold for proteasomal targeting by ubiquitin?

One ubiquitin molecule

Two ubiquitin molecules

At least four ubiquitin units

Any ubiquitin-linked protein

Q11. Which PTM is functionally irreversible and essential for blood coagulation?

Ser phosphorylation

Glu γ-carboxylation to Gla

Lys acetylation

Asn glycosylation

Q12. What chemical logic underlies protein splicing by inteins?

ATP-dependent peptide hydrolysis

Radical-mediated backbone cleavage

Thioester intermediates followed by acyl shift

Metal-catalyzed oxidation

Q13. Why is GFP chromophore formation classified as a posttranslational modification?

It requires external enzymes

It rearranges the peptide backbone after folding

It involves proteolytic cleavage

It occurs before translation

Q14. What is the functional role of the MIO (methylene-imidazolone) group?

Structural stabilization of collagen

Electrophilic cofactor for ammonia-lyase reactions

Metal ion chelation

Signal peptide cleavage

Q15. Posttranslational modifications occur only in eukaryotic cells.

True

False

Q16. Only 5 of the 20 standard amino acids are known to undergo posttranslational modification.

True

False

Q17. Phosphorylation typically introduces a negative charge that can alter protein conformation and interactions.

True

False

Q18. Ubiquitylation always leads to proteasomal degradation.

True

False

Q19. Incomplete glycosylation at multiple steps is a major source of glycoprotein heterogeneity.

True

False

Q20. Prenylation is reversible through dedicated cellular enzymes.

True

False

Q21. Histone tail modifications can act combinatorially to regulate gene expression.

True

False

Q22. Four ubiquitin molecules are generally sufficient to signal proteasomal degradation.

True

False

Q23. All posttranslational modifications are reversible.

True

False

Q24. Controlled proteolysis is considered a form of posttranslational modification.

True

False

Q25. Protein splicing requires external proteases to excise inteins.

True

False

Q26. GFP fluorescence arises only after the protein has folded into its native structure.

True

False

Q27. The MIO cofactor is genetically encoded as a nonstandard amino acid.

True

False

Q28. Reversibility of a PTM often correlates with its role in signaling rather than structural commitment.

True

False