Primary Structure of Proteins

Primary Structure: Definition & Basics - Amino Acid Chain Gang

• Definition: The linear sequence of amino acids in a polypeptide chain, determining the protein's identity.
• Peptide Bond: Amino acids are covalently linked by peptide bonds ($CONH$).
  • Formed via a dehydration reaction between the α-carboxyl group of one amino acid and the α-amino group of the next.
• Termini & Directionality:
  • N-terminus (Amino-terminus): The start of the chain, possessing a free amino group (-$NH_2$).
  • C-terminus (Carboxyl-terminus): The end of the chain, possessing a free carboxyl group (-$COOH$).
  • Read from N → C terminus.

⭐ The primary structure dictates all higher levels of protein structure (secondary, tertiary, quaternary) and thus its ultimate biological function. This sequence is genetically determined by DNA.

Peptide Bond: Characteristics - The Backbone's Blueprint

• Partial Double Bond Character:
  • Due to resonance, the C-N peptide bond has ~40% double bond character.
  • This makes it rigid and planar.
• Consequences of Rigidity:
  • Restricted Rotation: No rotation around the peptide bond (C-N axis) itself.
  • Trans Configuration Favored:
    • Adjacent $\alpha$-carbons ($C_{\alpha}$) are usually trans to minimize steric hindrance between R-groups.
    • Exception: Proline (X-Pro bonds) can adopt cis configuration more readily.
• Backbone Flexibility:
  • Rotation is permitted around bonds connected to the $\alpha$-carbon ($C_{\alpha}$):
    • N-$C_{\alpha}$ bond: Angle $\phi$ (phi).
    • $C_{\alpha}$-C bond: Angle $\psi$ (psi).
  • These $\phi$ and $\psi$ angles (Ramachandran angles) determine the polypeptide chain's conformation.

⭐ The peptide bond has approximately 40% double bond character due to resonance, making it rigid and planar.

Sequence Determination: Methods - Cracking the Protein Code

Crucial for protein function, evolution, & disease understanding.

• N-Terminal Analysis:
  • Sanger's Reagent (FDNB): Identifies N-terminal AA. Forms DNP-AA. Destroys peptide.
  • Edman Degradation (PITC): Sequential N-terminal AA ID. Phenylisothiocyanate (PITC) reacts with N-terminus, cleaved as PTH-AA. Peptide intact for next cycle.

    ⭐ Edman degradation allows sequential N-terminal AA removal and identification.

    ![Edman degradation process diagram](https://ylbwdadhbcjolwylidja.supabase.co/storage/v1/object/public/notes/L1/Biochemistry_Protein_Structure_and_Function_Primary_Structure_of_Proteins/a8f28ea1-ee7a-456e-912b-8ee866c1a9b8.png)

• C-Terminal Analysis:

  • Carboxypeptidases: Cleave C-terminal AAs.
    • A: Most AAs (not Arg, Lys, Pro).
    • B: Arg, Lys.
    • Y: Most AAs.

• Specific Cleavage (Enzymatic): Fragments protein.

  • Trypsin: Cleaves at C-side of Lys (K), Arg (R) (not if Pro follows). 📌 Try-K/R.
  • Chymotrypsin: Cleaves at C-side of Phe (F), Tyr (Y), Trp (W) (aromatics) (not if Pro follows). 📌 Chymo-F/Y/W.

• Mass Spectrometry (MS): Modern, rapid, sensitive for sequencing & ID.

Clinical Correlation: Primary Structure Defects - When Sequences Go Wrong

• Genetic mutations alter amino acid sequences, causing defective proteins and disease.
• Key Examples:
  • Sickle Cell Anemia (HbS): $\beta$-globin gene mutation: Glu6Val (Glutamic acid $\rightarrow$ Valine at position 6).

    ⭐ Sickle cell anemia is caused by a single amino acid substitution (Glu$\rightarrow$Val at position 6) in the $\beta$-globin chain of hemoglobin.

  • Cystic Fibrosis (CFTR): $\Delta$F508 (Phenylalanine deletion at 508) in CFTR protein.
  • Phenylketonuria (PKU): Mutations in Phenylalanine Hydroxylase (PAH) enzyme.
• Substitutions Impact:
  • Conservative: Similar amino acid; minor functional change.
  • Non-conservative: Dissimilar amino acid; major functional disruption.

High‑Yield Points - ⚡ Biggest Takeaways

• Primary structure: The linear sequence of amino acids linked by peptide bonds.
• Peptide bond: Exhibits partial double bond character, resulting in rigidity and planarity.
• Genetic determination: The amino acid sequence is encoded by DNA.
• Structural determinant: Dictates all higher levels of protein structure and thus function.
• Directionality: Defined by the N-terminus (amino) and C-terminus (carboxyl).
• Clinical significance: Sequence changes (e.g., sickle cell anemia: Glu⁶Val) can lead to disease.