CHEM 114A: Chapter 4 - Lecture 4

Calculating the Isoelectric Point (pI) of a Peptide

• Goal : find the pH at which the peptide’s net charge = 0.
• Recipe applied in the example peptide (5 ionisable groups: N-terminus, C-terminus + 3 side chains)
  • 1️⃣ List every ionisable group and write its $pK_a$ value.
  • N-terminus
  • C-terminus
  • Side-chain 1: $pK_a = 3.60$ (written as 3.66 in the narration)
  • Side-chain 2: $pK_a = 4.25$
  • Side-chain 3: $pK_a = 6.00$
  • Additional groups at $pK<em>a = 9.20$ and $pK</em>a = 12.48$ (C-terminus & N-terminus respectively).
  • 2️⃣ Draw the fully protonated structure (assume $pH = 0$); sum the charges → +2.
  • 3️⃣ Raise the pH stepwise in order of ascending $pK_a$, de-protonating one group at a time and updating the formal charge.
  • After $pK_a = 3.60$ → charge = +1.
  • After $pK_a = 4.25$ → charge = 0.
  • After $pK_a = 6.00$ → charge = –1.
  • After $pK_a = 9.20$ → charge = –2.
  • After $pK_a = 12.48$ → charge = –3.
  • 4️⃣ Locate the charge-0 species (here at pH 4.25).
  • 5️⃣ Average the two $pK_a$ values that bracket the neutral species:
    $pI = \frac{3.66 + 4.25}{2} = 4.80$
  • Pointer : general formula
    $\boxed{pI = \dfrac{pK<em>a\,(\text{just below 0 charge}) + pK</em>a\,(\text{just above 0 charge})}{2}}$

Resonance and Planarity of the Peptide Bond

• The O–C–N atoms form a delocalised $\pi$ system.
  • Electrons from the carbonyl C=O can shift to form C=N between the carbon and amide N, giving structures A & B.
• Consequences (≈ 40 % double-bond character):
  • C–N (peptide) bond length ≈ $0.13\,\text{Å}$ shorter than a regular $C_\alpha–N$ single bond → stronger and more rigid.
  • C=O bond length ≈ $0.02\,\text{Å}$ longer than in ketones/aldehydes.
  • The six atoms $O–C–N–C_\alpha–C–O$ lie in a single plane (peptide plane).
  • High rotational barrier ≈ 88\,\text{kJ mol^{–1}}; free rotation is essentially prevented.

Cis–Trans Isomerism Around the Peptide Bond

• Because rotation is hindered, we describe the relative positions of the two adjacent $C_\alpha$ atoms as cis or trans.
  • Trans (≈ 99.9 % of bonds) : $R<em>{i}$ and $R</em>{i+1}$ on opposite sides → minimal steric clash.
  • Cis : $R$ groups on same side → severe steric hindrance unless both residues are tiny (e.g.
    Gly–Gly).
• Energetic preference driven purely by steric repulsion between side chains.

Special Case : Proline-Containing Bonds

• Proline’s side chain forms a ring with the backbone N, inserting bulk both above & below the plane.
• Both cis and trans have steric clashes, but cis is slightly less strained, hence appreciably populated (up to ~10 %).
• Biological significance :
  • Cis-Pro lines often act as molecular “hinges”, forcing a 180° turn (β-turn) in the polypeptide chain.
  • Enzymes (peptidyl-prolyl isomerases) catalyse inter-conversion, regulating folding & signalling pathways.

Extended Polypeptide View

• In an all-trans backbone the peptide planes stack like tiles; side chains alternate above/below plane.
• Inserting a cis-Pro instantly redirects the chain trajectory.
• Sets the stage for secondary/tertiary structure discussions in later chapters.

Post-Translationally Modified (Uncommon) Amino Acids

• Hydroxyproline
  • Enzyme : prolyl hydroxylase (post-translational).
  • ~4 % of all animal AAs; ≈13.5 % of collagen.
  • Adds stability via additional H-bonding → crucial for connective tissue integrity.
• 3-Methyl-histidine
  • Found in actin & myosin; concentration rises after muscle injury → biomarker of muscle breakdown.
• ε-N-Acetyl-lysine
  • Histone & non-histone proteins.
  • Neutralises Lys positive charge → weakens DNA binding and modulates gene expression (epigenetics).
• Take-home : PTMs expand chemical functionality beyond the genetic 20-AA alphabet.

Green Fluorescent Protein (GFP) & Engineered Fluorophores

• Origin : Aequorea victoria jellyfish; peak emission $\lambda_{max} = 508\,\text{nm}$.
• Robert Tsien (UCSD) received 2008 Nobel Prize for elucidation & optimisation.
• Intrinsic fluorophore formed by Ser-Tyr-Gly (positions 65-67) via spontaneous cyclisation + oxidation.
  1. Ser carbonyl attacks Gly N (nucleophilic acyl substitution) → five-membered ring.
  2. Concomitant oxidation extends $\pi$-conjugation.
• Mutation S65T
  • Bulkier Thr retains hydroxyl but enhances stability → brighter and shifts spectrum.
• Palette expansion : mutagenesis around chromophore produced blue, cyan, yellow, red FPs.
• Applications
  • Multicolour imaging of protein–protein interactions in live cells.
  • Transgenic organisms (e.g.
    mice) that fluoresce under UV in thin tissues (tail, ears, eyes, nose).

Bioactive Small-Molecule AA Derivatives (Neuro- / Endo-crine Roles)

• GABA (γ-aminobutyric acid)
  • Derived from Glu; chief inhibitory neurotransmitter, mood regulation.
• Histamine
  • Decarboxylated His; mediates inflammatory & allergic responses.
• Dopamine
  • Hydroxylated & decarboxylated Tyr; reward, motor control.
• Thyroxine (T4)
  • Iodinated Tyr derivative; thyroid hormone regulating metabolism.

Glutathione (GSH) – A Non-Canonical Tripeptide

• Composition : $\gamma$-Glu–Cys–Gly.
  • Isopeptide bond : side-chain (γ-carboxyl) of Glu linked to Cys N.
• Active moiety : Cys thiol \ce{–SH}.
• Dimerisation
  $2\,GSH \xrightarrow[dehydration]{oxidation} GSSG$ (disulfide)
• Biological roles
  • Universal cellular redox buffer; oxidised/reduced ratio gauges oxidative stress.
  • Detoxifies peroxides & electrophiles, converting itself from GSH to GSSG (coupled reduction of target).
  • Maintains proteins’ Cys residues in reduced state; can reduce inappropriate inter-/intra-chain disulfides.
    • Example : reduces disulfide bridges in bovine insulin, destroying its active conformation.

Preview – Connection to Upcoming Structure Chapters

• Resonance-imposed planarity + cis/trans constraints define the backbone torsion angles $\phi$ and $\psi$ (Ramachandran space).
• Proline’s rigidity and cis propensity act as conformational “gatekeepers”.
• Disulfide chemistry (internal, between chains, or with glutathione) crucial for stabilising tertiary/quaternary structure.
• PTMs and non-standard amino acids add yet another layer of structural & functional diversity.