Proteins – Comprehensive Study Notes

21st-Century Context – Infectious Prions

• Prions are infectious proteins that lack DNA/RNA but propagate by inducing misfolding of normal prion protein (PrP(^{C}$) into the pathogenic form PrP(^{Sc}$).
• Structural difference:
◦ PrP(^{C}$≈ "tiny spheres" (mainly (\alpha$-helical).
◦ PrP(^{Sc}$≈ "cubes" (rich in (\beta$-sheets).
• Misfolding initiates in the disordered N-terminal region; conversion studied with X-ray crystallography and nanobody scaffolds (Abskharon et al., 2014; DOI:10.1021/ja407527p).
• Significance: understanding early misfolding events can guide drug design against spongiform encephalopathies (e.g. mad-cow, CJD).
• Potential therapies discussed: stabilising correct fold, blocking PrP(^{Sc}$–PrP(^{C}$ interaction, using nanobodies or small molecules, promoting clearance.

1. Introduction to Proteins

• Complex organic macromolecules composed mainly of C, H, O, N; S and P may occur.
• Contribute >$50\%$ of dry cell mass; participate in virtually every biological process (enzymes, transport, structure, signalling, defence, movement, storage).
• Functional form = one or more polypeptides folded into specific 3-D conformations (ribbon model shown).

2. Amino Acids (monomers)

• General structure: central ((\alpha$)-C attached to –H, –NH(<em>2$), –COOH, and variable R-group. • 20 naturally occurring amino acids; R-group dictates chemical behaviour and ultimately protein structure/function. • Categories by R-group: ◦ Non-polar, neutral → hydrophobic. ◦ Polar, neutral → hydrophilic. ◦ Polar, acidic → extra –COOH (overall (-1 charge). ◦ Polar, basic → extra –NH(2 (overall (+1 charge).
• Unique residues: cysteine (–SH) forms disulphides; proline induces kinks; glycine provides flexibility.
• At physiological pH each amino acid exists mainly as a zwitterion:
◦ \text{–NH}2 + H^+ \rightarrow \text{–NH}3^+
◦ \text{–COOH} \rightarrow \text{–COO}^- + H^+
• Zwitterionic nature → buffering capacity (Fig 2.3).
• Solubility: generally water-soluble (ionic), insoluble in non-polar solvents.

3. Peptide Bonds & Polypeptides

• Condensation (dehydration) between –NH(2 of one aa and –COOH of another → peptide bond + $H</em>2O$ (enzyme-catalysed).
• Dipeptide retains N-terminus (free –NH(_3^+$) and C-terminus (free –COO(^-$) → allows chain elongation.
• Repetition yields polypeptide (primary structure).
• Hydrolysis: by proteases or strong acid.
• Isomer possibilities:
◦ For (n$distinct residues used once each →$n!$permutations (e.g.$5! = 120).
◦ For polypeptide length (p drawn from pool of (n$types with replacement →$n^p possibilities.
• Backbone = repeating N–C(\alpha–C(O) atoms; side chains extend outward.

4. Stabilising Interactions Within/Between Polypeptides

• Four major non-peptide interactions determine higher-order structure:

1. Ionic bonds: between oppositely charged groups ((\text{–NH}_3^+$⇄ (\text{–COO}^-$) on side chains or termini; pH-dependent.
2. Hydrogen bonds: H attached to electronegative atom (O/N) attracted to another O/N; individually weak, collectively stabilising (e.g. (\alpha-helix).
3. Hydrophobic interactions: clustering of non-polar R-groups away from water → hydrophobic core; major driving force in globular folding & quaternary assembly.
4. Disulphide bridges: covalent –S–S– between two cysteines (oxidation); intra- or inter-chain; broken only by reducing agents.

5. Levels of Protein Structure

• Primary: linear aa sequence (peptide bonds).
• Secondary: local regular folding of backbone into (\alpha-helix or (\beta-pleated sheet; stabilised by backbone H-bonds (C=O···H–N).
• Tertiary: overall 3-D shape of single polypeptide; maintained by ionic, H-bond, hydrophobic, and disulphide interactions among side chains.
• Quaternary: spatial arrangement of multiple subunits; same four interaction types (hydrophobic main driver).
◦ Example: haemoglobin (4 subunits).
• Folding facilitated by chaperonins (e.g. GroEL/GroES in E. coli) which provide isolated hydrophilic cavity for spontaneous folding.

6. Details of Secondary Structures

• (\alpha-Helix: right-handed coil, 3.6 residues per turn; H-bond between residue (n$C=O and residue (n+4$ N–H; R-groups project radially; common in enzymes/antibodies.
• (\beta-Sheet: extended strands laid side-by-side; H-bonds between adjacent chains; strands can be parallel (same N→C) or antiparallel (opposite); impart tensile strength (e.g. silk fibroin).

7. Classification Schemes

• By composition:
◦ Simple proteins – only amino acids (albumin, collagen).
◦ Conjugated proteins – amino acids + non-protein prosthetic group (haem of haemoglobin, FAD of flavoproteins, carbohydrate of glycoproteins).
• By structure:
◦ Fibrous – long, water-insoluble, mainly secondary structure, structural roles (collagen, keratin).
◦ Globular – compact, water-soluble, complex folding, metabolic roles (enzymes, antibodies, haemoglobin).
• Key comparative features (Table 7.1): sequence regularity, solubility, stability, length variability, etc.
• By function – transport, catalysis, signalling, defense, etc.

8. Haemoglobin – Structure–Function Correlation

• Tetrameric globular protein (quaternary): \alpha2\beta2.
• Each subunit ≈ 141–146 aa and bears a prosthetic haem (protoporphyrin IX + Fe(^{2+}).
• Iron coordinated to histidine on one face; other face reversibly binds (O2. • Cooperative binding: O(2 binding to one haem induces conformational change → increases affinity of remaining sites (sigmoidal saturation curve).
• Hydrophobic core + outward hydrophilic residues → aqueous solubility inside erythrocytes.
• Compactness allows high packing density in RBCs for efficient transport.

9. HIV-1 Protease – Enzymatic Example

• Homodimer of two identical 99-aa chains; only one active site formed at dimer interface (four-stranded antiparallel (\beta-sheet).
• Secondary elements: 1 (\alpha-helix + 2 antiparallel (\beta-sheets per monomer.
• Quaternary stabilisation by hydrophobic packing; crucial for catalytic activity.
• Clinical relevance: target of antiretroviral protease inhibitors.

10. Factors Affecting Protein Structure – Denaturation & Renaturation

• Denaturation = loss of secondary, tertiary, quaternary structure → loss of biological activity; primary remains intact.
• Physical/Chemical agents:
◦ Temperature: ↑KE above optimum breaks weak interactions; egg-white coagulation is example; high fever can be lethal.
◦ pH: alters ([H^+] → changes ionisation of acidic/basic R-groups → disrupts H- and ionic bonds.
▪ Small pH shifts reversible; large shifts irreversible.
◦ Organic solvents: flip hydrophobic regions outward.
◦ Heavy-metal cations (Hg(^{2+}$, Pb(^{2+}$): bind –COO(^- groups, neutralise charges, cause precipitation.
◦ Reducing agents: break disulphide bridges.
• Denatured proteins often aggregate due to exposed hydrophobic patches; depicted in Fig 10.1.
• Renaturation possible if denaturing agent removed before extensive aggregation (Fig 10.2).
• Overall hierarchy: primary sequence dictates folding → folding determines function; environmental perturbations disrupt interactions → loss of function.

Mathematical & Statistical References

• Number of peptide isomers using each amino acid once: n!.
• Number of possible polypeptides of length p$from (n$ amino acids with repetition: $n^p$.
• Dry mass contribution of proteins: >$50\%$.

Ethical & Biomedical Implications

• Prion research informs strategies to combat fatal neurodegenerative diseases lacking cures; underscores need for rigorous control of animal feed & surgical instruments.
• HIV protease inhibitors highlight structure-guided drug design.
• Haemoglobinopathies (e.g. sickle-cell from single aa change) illustrate importance of precise primary structure—medical, evolutionary, and genetic-counselling relevance.

Conceptual Checks (Self-Test Prompts)

• Label N-terminus, C-terminus, side chains, peptide bonds on a polypeptide diagram.
• Draw the tripeptide Met-Gly-Ala, indicating peptide bonds and termini.
• Complete comparison table of four structural levels: bonds involved, number of chains, bonding partners.
• List and explain at least seven denaturing factors (temperature, pH, organic solvents, heavy metals, reducing agents, radiation, mechanical agitation).
• Describe how hydrophobic interactions drive globular folding and quaternary assembly.

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