Comprehensive Notes on Amino Acids, Peptides, and Proteins

Overview of Protein Functions and Diversity

• Proteins include catalysts (enzymes), structural fibers (keratin, collagen), transporters (hemoglobin), antibodies, hormones, light-harvesting complexes, toxins, etc.

• A single cell may contain thousands of distinct proteins; diversity arises from different linear sequences of 20 common α-amino acids.

• Example functions

  

  • Firefly luciferin–luciferase light production (ATP-dependent).

  • Oxygen transport by erythrocyte hemoglobin.

  • Keratin in hair, nails, wool, rhinoceros horn.

Four Organizing Principles

• All living organisms build proteins from the same 20 amino acids.

• Each amino acid has a side chain (R group) with distinctive chemistry.

• Amino acids join via peptide bonds → linear sequence = primary structure.

• Individual proteins can be purified because unique sequences confer unique chemical/functional properties; sequences also trace evolutionary relationships.

3.1 Amino Acids

Common Structural Features (Fig. 3-2)

• General formula: \text{H}_3\text N^{+}–\text{CH}(\text R)–\text{COO}^{-} at pH 7.

• All are α-amino acids ➜ amino & carboxyl on same carbon (α-carbon).

• "Residue" = amino acid minus elements of \text H_2\text O after peptide bond.

Chirality & Stereoisomerism (Fig. 3-3, 3-4)

• Except glycine (where the R group is hydrogen, making two identical groups attached to the {\alpha} -carbon), the {\alpha} -carbon in amino acids is chiral. chiral carbon: carbon atom bonded to four different groups.

• This chirality leads to the existence of two non-superimposable mirror-image forms called D & L enantiomers.

• Biological proteins almost exclusively use L-amino acids (comprising more than 99% of amino acids found in proteins).

• While chiral compounds do rotate plane-polarized light (a property known as optical activity), the D/L designation refers to the absolute configuration of the molecule's atoms in space, specifically relative to the stereochemistry of L-glyceraldehyde, and not directly to the direction or magnitude of optical rotation.

Carbon Nomenclatures

• Side-chain carbons: β, γ, δ … outward from α.

• Alternative numeric system (C-1 as carboxyl C) used when Greek ambiguous.

Classification by R Groups (Table 3-1, Fig. 3-5)

Non-polar, Aliphatic

• Gly, Ala, Val, Leu, Ile, Met, Pro.

• Hydrophobic clustering stabilizes protein core.

• Gly provides flexibility; Pro imposes rigidity (cyclic secondary amino).

Aromatic

• Phe, Tyr, Trp ➜ relatively hydrophobic.

• Tyr OH can H-bond & undergo phosphorylation.

• UV absorbance peaks near 280\,\text{nm} (Tyr & Trp > Phe) ➜ used to estimate protein concentration (Lambert–Beer law A = \varepsilon c l).

Polar, Uncharged

• Ser, Thr (OH), Cys (SH), Asn, Gln (amide).

• Cys can oxidize → disulfide bond (cystine) strengthening protein structure.

Positively Charged (Basic)

• Lys (\text{p}K_R \approx 10.53), Arg (12.48), His (6.00 ‑ near neutrality ➜ enzymatic acid–base catalyst).

Negatively Charged (Acidic)

• Asp (\text{p}K_R = 3.65), Glu (4.25) – deprotonated at pH 7.

Uncommon & Post-translationally Modified Amino Acids (Fig. 3-8)

• Examples: 4-Hydroxyproline (collagen), γ-Carboxyglutamate (prothrombin), desmosine (elastin cross-link).

• Selenocysteine & pyrrolysine inserted co-translationally via recoded stop codons.

• Reversible modifications regulate activity (phosphorylation, methylation, acetylation, ADP-ribosylation, etc.).

• Ornithine & citrulline: urea cycle intermediates, not protein residues.

Acid–Base Properties & Titration

• In water, amino acids exist mainly as zwitterions.

• Diprotic acids (for simple aa): \mathrm{p}K1(\alpha\text{–COOH}) \approx 2.0; \mathrm{p}K2(\alpha\text{–NH}_3^+) \approx 9.5.

• Henderson–Hasselbalch: \text{pH}=\text{p}K_a+\log\dfrac{[A^-]}{[HA]}.

• Titration of glycine (Fig. 3-10):

  • Buffering at \text{p}K1=2.34 & \text{p}K2=9.60.

  • Isoelectric point \text{pI}=\dfrac{2.34+9.60}{2}=5.97 ➜ net charge 0.

• Amino acids with ionizable R groups display three stages & three \text pK values (examples: glutamate, histidine; Fig. 3-12). \text{pI}{\text{acidic}} \approx \frac{\text{p}K1+\text{p}KR}{2}; \text{pI}{\text{basic}} \approx \frac{\text{p}K2+\text{p}KR}{2}.

Summary Highlights

• 20 common L-α-amino acids are the “alphabet” of proteins.

• R-group chemistry dictates solubility, reactivity, charge.

• Knowledge of structures, abbreviations, pKa and classification is foundational for biochemistry.

3.2 Peptides and Proteins

Peptide Bond Formation (Fig. 3-13)

• Condensation reaction: \text{COO}^- + \text{NH}3^+ \;\xrightarrow{\;\Delta H2O\;}\; peptide bond \text{–CO–NH–} + \text H_2\text O.

• Thermodynamically favors hydrolysis; biosynthesis uses activated carboxyl (tRNA) + ribosomal catalysis.

• Di-, tri-, oligo- (< ~20), poly- (> ~20–50) peptides.

Directionality & Terminology (Fig. 3-14)

• Sequence written N-terminal → C-terminal.

• Example pentapeptide: Ser-Gly-Tyr-Ala-Leu (SGYAL).

Stability & Ionization of Peptides (Fig. 3-15)

• Peptide bonds have t_{1/2} \approx 7\,\text{yr} in cells (high activation barrier to hydrolysis).

• Only terminal \alpha-NH$_3^+$ & \alpha-COO$^- ionize; internal groups engaged in peptide bonds.

• Net charge ↔ pH ➜ exploited in separations.

Size Range & Biological Examples (Tables 3-2, 3-3)

• Small peptides with potent activity: oxytocin (9 aa), thyrotropin-releasing factor (3 aa), amanitin toxin.

• Proteins: cytochrome c (104 aa), chymotrypsinogen (245), hemoglobin (574 in 4 subunits), titin (~ 27,000 aa, M_r \approx 3\times10^6).

• Average residue mass ≈ 110\,\text{Da} ➜ \text{No. residues} \approx \dfrac{M_r}{110}.

Quaternary & Conjugated Proteins

• Multisubunit proteins held non-covalently; identical subunits → oligomeric, minimal repeating unit = protomer.

• Conjugated proteins possess non-aa prosthetic groups (Table 3-4): lipids, carbohydrates, phosphate, heme, flavin, metals (Fe, Zn, Ca, Cu, Mo).

Summary Points

• Peptide bond defines primary structure; N→C orientation mandatory.

• Proteins vary from few to > 10⁴ residues; may have covalent or non-covalent prosthetic groups.

3.3 Working with Proteins

General Strategy

1. Source: cells/tissues → disrupt to crude extract.

2. Fractionation exploiting solubility, size, charge, binding.

3. Progressive purification monitored by assay & specific activity.

Early Steps

• Salting out with \text{(NH}4)2\text{SO}_4; different proteins precipitate at different ionic strengths.

• Dialysis removes small solutes through semipermeable membrane.

Column Chromatography (Fig. 3-16, 3-17)

• Stationary phase (matrix) vs mobile phase (buffer).

• Ion-exchange: cation exchangers (negatively charged resin) bind + proteins; elute by salt or pH gradient.

• Size-exclusion (gel-filtration): porous beads; large proteins elute first.

• Affinity: matrix-bound ligand specifically binds target protein; elute by free ligand or salt. Engineered tags (His₆, GST, etc.) facilitate single-step purification.

• HPLC: high pressure + fine matrix ➜ high resolution, fast.

Monitoring Purification

• Electrophoresis in polyacrylamide gels:

  • With SDS ➜ uniform q/m, separation by size; visualize with Coomassie blue (Fig. 3-18, 3-19).

  • Molecular weight from log (M_r) vs mobility plot.

  • Isoelectric focusing: separation by pI in pH gradient (Fig. 3-20).

  • 2-D electrophoresis = IEF (1st) + SDS-PAGE (2nd) ➜ resolves thousands of proteins (Fig. 3-21).

Enzyme Assays & Purification Table (Table 3-5)

• Activity: μmol substrate converted · min⁻¹.

• Specific activity: units · mg⁻¹ – increases with purity.

• Purification factor = \dfrac{\text{final specific activity}}{\text{initial}}; yield = \dfrac{\text{final total activity}}{\text{initial}}.

Summary

• Combine inexpensive bulk methods first, high-resolution methods later.

• SDS-PAGE & specific activity benchmarks guide purification.

3.4 Protein Structure – Primary Structure

Hierarchy (Fig. 3-23)

• Primary (sequence) → Secondary (α-helix, β-sheet) → Tertiary (3-D fold) → Quaternary (subunit assembly).

Functional Importance of Sequence

• Unique sequence → unique 3-D structure → unique function.

• Mutations (single residue to deletions) underlie many diseases (e.g., sickle cell anemia, dystrophin in muscular dystrophy).

• Proteins are often polymorphic; conserved regions denote functional/structural importance.

Classical Sequencing Tools

• Edman degradation: stepwise N-terminal PITC labeling & cleavage.

• N-terminal labeling reagents: FDNB, dansyl, dabsyl chlorides (Fig. 3-25).

• Disulfide cleavage: performic acid oxidation; reduction with dithiothreitol or β-mercaptoethanol + alkylation (Fig. 3-26).

• Protease/chemical cleavage specificities (Table 3-6):

  • Trypsin: Lys/Arg (C-side); Chymotrypsin: Phe/Tyr/Trp (C);

  • CNBr: Met (C); V8 protease: Asp/Glu (C), etc.

Synthetic Peptide Chemistry (Merrifield, Fig. 3-30)

• Solid-phase synthesis using Fmoc strategy.

• Repetitive cycle: deprotect → activate incoming aa → couple → wash.

• Direction: C-terminus anchored to resin; synthesis proceeds C→N (reverse of biosynthesis).

• Practical length ≤ ~100 residues; peptides can be ligated for larger proteins.

Mass Spectrometry & Proteomics

• MALDI-MS & ESI-MS move macromolecules to gas phase with minimal fragmentation.

• m/z measurement via TOF or Orbitrap ➜ accurate M_r$$ (Fig. 3-28).

• Tandem MS (MS/MS): protease digest → select one peptide → collide & fragment → deduce sequence from mass differences (Fig. 3-29).

• LC-MS/MS enables whole-proteome identification & quantification within ~1 h.

Bioinformatics & Evolution

• Consensus sequence: most frequent residue per position (Box 3-2); sequence logos visualize conservation.

• Homologous proteins: paralogs (within species), orthologs (between species).

• Horizontal gene transfer complicates trees.

• Signature sequences identify clades (e.g., 12-aa insertion in EF-1α/EF-Tu of Archaea/Eukarya; Fig. 3-32).

• Multiple conserved proteins (e.g., 120 bacterial proteins) produce calibrated phylogenetic trees (Fig. 3-33).

Summary Bullets

• Primary structure can be obtained from DNA or MS; informs folding, function, regulation, location, and evolutionary history.

• Chemical & enzymatic tools allow manipulation, modification, synthesis of peptides for research & therapeutics.