📘 Overview (Page 1)

This lecture covers four big themes:

1. Amino acid chemical reactivity & PTMs
2. Protein size
3. How protein size is determined
4. Protein sequence determination

This sets up the link between chemistry → structure → analytical methods

🧪 Chemical Reactivity of Amino Acids (Pages 2–24)

Page 2 – Nucleophiles vs Bases

• Most protein chemistry occurs in water, ~neutral pH
• Reactive groups = nucleophiles
• Key distinction:
  • Basicity → affinity for H⁺
  • Nucleophilicity → attacks electrophilic atoms
• A group can be a strong nucleophile without being a strong base (very important later for Cys, Met!)

The image lists specific side chains, enzymes they appear in, and reaction intermediates.

Pages 3–4 – Serine & Threonine

• Side chain: –OH
• Weak nucleophiles unless deprotonated (–O⁻)
• Common PTMs:
  • Phosphorylation
  • O-glycosylation
  • Acetylation (rare)
• In enzymes, Ser reacts as a serine alkoxide

Page 4 (image): Catalytic triad 🧠

• Ser–His–Asp
• His abstracts a proton from Ser → Ser becomes a strong nucleophile
• Asp stabilizes His
• This explains why Ser can be reactive despite a high pKa

Page 5 – Phosphorylation

• Adds a di-anionic phosphate
• Causes local conformational changes
• Mostly Ser/Thr/Tyr in eukaryotes
• His and Asp phosphorylation common in bacteria/fungi
• Acts as a molecular switch

Page 6 – O-Glycosylation

• Occurs in ER + Golgi
• No strict consensus sequence
• Enzyme-controlled (glycosyltransferases)
• Structurally simpler than N-glycosylation

Pages 7–8 – Aspartate & Glutamate

• Carboxylates (–COO⁻) → negative charge
• Excellent metal ion ligands (Ca²⁺, Zn²⁺)
• Used in carbodiimide coupling (peptide synthesis)

Page 8 focuses on chemical discrimination between –COO⁻ and –COOH using selective reagents.

Also:

• Asp is the nucleophile in P-type ATPases (phosphorylated intermediate)

Page 9 – Asparagine & Glutamine

• Amide side chains → polar but inert
• Important PTMs:
  • N-glycosylation (Asn)
  • Gln–Lys crosslinks
  • Gln–Cys thioesters
• Can be deaminated → Asp/Glu

Page 10 – N-Glycosylation 🧬

• Initiated in ER membrane
• Consensus: Asn-X-Ser/Thr
• Highly branched & processed in Golgi
• Important for folding, stability, trafficking

Page 11 – Protein Cross-Linking

• Catalyzed by transglutaminases
• Gln–Lys isopeptide bonds
• Used industrially (e.g. plant-based meat 😄)
• Also important biologically (clotting, ECM)

Pages 12–14 – Lysine Reactivity

• Primary amine, positively charged
• Strong nucleophile ONLY when deprotonated
• Reactivity ↑ at high pH

Applications:

• TNBS assay → counts Lys residues (λ = 367 nm)
• Acetylation (histones)
• Carbamylation → homocitrulline (uremia marker)

Page 15 – Alkylation of Lys & Arg

• Methylation & acetylation regulate DNA binding
• Histone code logic
• Other N-acylations:
  • Biotinyl
  • Lipoyl
  • Ubiquityl (protein degradation)

Page 16 – Arginine

• Guanidinium group
• Charge delocalized → chemically inert
• Methylation common (regulation)

Page 17 – Histidine ⭐

• Imidazole ring
• pKa ~6–7 → perfect for acid-base catalysis
• Strong metal ligand
• Central residue in many enzymes
• Can be protonated/deprotonated under physiological conditions

Page 18 – Tyrosine

• Phenolic side chain
• Phosphorylation important in signaling
• Less reactive than Ser/Thr but higher regulatory impact

Page 19 – Tryptophan

• Indole ring
• Sensitive to oxidation
• Limited reactivity
• Strong fluorescence relevance (later courses)

Page 20 – Methionine

• Thioether sulfur
• Cannot be protonated
• Strong nucleophile at low pH
• Easily oxidized
• Often removed after translation

Pages 21–23 – Cysteine 🧨

• Most reactive side chain
• pKa ≈ 8–8.5, but environment lowers it
• Reactive even at physiological pH
• Forms:
  • Disulfides
  • Sulfenic / sulfinic / sulfonic acids
• Metal binding (Zn²⁺ fingers!)

Page 23:

• DTNB (Ellman’s reagent) → counts free thiols
• Yellow NTB product (λ = 412 nm)

Page 24 – Disulfide Reduction

• TCEP reduces S–S bonds
• Stable, odorless alternative to DTT

🔪 Proteolysis & Sequencing (Pages 25–39)

Page 25 – Proteolytic Processing

• Zymogen activation
• Signal peptide removal
• Post-translational trimming

Page 26 – How to Determine Sequence

Methods:

1. Databases
2. DNA → translation
3. Protein digestion + MS/MS
4. Edman degradation

Page 27 – Enzymatic Cleavage Specificity

• Trypsin → C-term Lys/Arg
• Chymotrypsin → aromatics
• V8 → acidic residues
• Asp-N → N-term Asp
• Thermolysin → N-term hydrophobic

Pages 28–29 – Chemical Cleavage

• Non-enzymatic specificity
• Page 29: Cys cyanylation → selective backbone cleavage

Pages 30–32 – Edman Degradation

• Sequential N-terminal removal
• pH ~9 coupling
• TFA cleavage
• PTH-AA identified by RP-HPLC

Limitations: blocked N-termini, large proteins

⚡ Mass Spectrometry & Fragmentation (Pages 36–39)

Pages 36–37 – Why MS/MS?

• MALDI & ESI are soft ionization
• No fragmentation → no sequence info
• Peptides with same mass ≠ same sequence
• CID, ECD, ETD introduce backbone cleavage

Page 38 – Fragmentation Nomenclature

• Roepstorff–Fohlman / Biemann
• CID vs ETD differences
• Fragment ions depend on cleavage site

Page 39 – b and y ions explained clearly 🔥

This is critical.

What breaks?

• Peptide bond
• Charge stays either on N-terminal fragment (b-ion) or C-terminal fragment (y-ion)

b-ions:

• Contain N-terminus
• Sequence grows from left → right
• b₂ = first two amino acids

y-ions:

• Contain C-terminus
• Sequence grows from right → left
• y₁ = last amino acid

Why both?

• Overlapping ladders → unambiguous sequencing
• Differences in m/z reveal residue masses

This is how MS/MS “reads” peptides.

📏 Protein Size & Shape (Pages 40–61)

Page 40 – Protein Size Range

• Insulin: ~5.8 kDa
• Rubisco: ~540 kDa
• Ribosome: ~2 MDa
• Myosin, actin filaments shown visually

Page 41 – Rules of Thumb

• Avg aa ≈ 110 Da
• Avg human protein ≈ 373 aa
• Titin = 33,423 aa (~3.7 MDa!)

Size determination methods listed.

Pages 42–44 – Ultracentrifugation & Electrophoresis

• Svedberg & Tiselius
• Revealed:
  • Oligomeric state
  • Shape
  • Homogeneity
• Basis for SDS-PAGE calibration

Pages 46–47 – Shape Models

• Random coil
• Rod
• Prolate / Oblate
• Globular proteins have rough surfaces

Pages 48–51 – SDS-PAGE

• SDS denatures proteins
• ~1 SDS per 2 aa → uniform charge
• Rod-like migration
• Log(Mw) vs mobility linear
• Staining:
  • Coomassie
  • Silver
  • Western blot

Pages 52–56 – Mass Spectrometry

• MALDI-TOF → mostly singly charged
• ESI → charge ladders
• Deconvolution → exact mass
• Charge ≈ 1 per kDa

Pages 57–60 – Size Exclusion Chromatography

• Separation by hydrodynamic radius
• Native proteins elute earlier
• Denatured proteins appear larger
• Calibration with standards

Page 61 – Method Comparison

• SEC, MS, SDS-PAGE, ultracentrifugation compared
• Each measures a different physical property

Page 62 – End

Summary slide / transition

🧠 Key Take-Home Messages

• Reactivity ≠ charge ≠ pKa
• Protein chemistry depends on microenvironment
• PTMs regulate function
• MS/MS + b/y ions = modern sequencing
• Size depends on method and shape