Protein Chemistry
📘 Overview (Page 1)
This lecture covers four big themes:
- Amino acid chemical reactivity & PTMs
- Protein size
- How protein size is determined
- 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:
- Databases
- DNA → translation
- Protein digestion + MS/MS
- 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
Quiz
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