Advanced Topics

Inteins and Protein Splicing

Summary

Inteins are autocatalytic protein domains that excise themselves from precursor polypeptides while ligating the flanking exteins. This protein splicing mechanism is now a critical tool in biotechnology.

Key Points

  • 1Self-splicing through four-step autocatalytic mechanism
  • 2Split inteins enable trans-splicing of separate polypeptides
  • 3Likely evolved as selfish genetic elements
  • 4Critical tools for protein purification and semi-synthesis

Inteins represent a remarkable case of proteins that process themselves, with applications ranging from basic research to biotechnology.

What Are Inteins?

Inteins (Internal proteins) are self-splicing protein elements:

  • Excise themselves from a precursor polypeptide
  • Ligate the flanking sequences (exteins) with a native peptide bond
  • Require no external factors or energy sources
  • Function entirely through autocatalysis

    • The Protein Splicing Mechanism

    Protein splicing proceeds through a conserved four-step mechanism:

    Step 1: N-O/S Acyl Shift

  • The first residue of the intein (Cys or Ser) attacks the preceding peptide bond
  • Forms a (thio)ester intermediate
  • Linearizes the junction

    • Step 2: Transesterification

  • The first residue of the C-extein (Cys, Ser, or Thr) attacks the (thio)ester
  • Creates a branched intermediate
  • Transfers the N-extein to the C-extein residue

    • Step 3: Asparagine Cyclization

  • A conserved Asn at the intein C-terminus cyclizes
  • Forms a succinimide ring
  • Cleaves the peptide bond to release the intein

    • Step 4: O/S-N Acyl Shift

  • The (thio)ester bond spontaneously rearranges
  • Forms a stable amide (peptide) bond
  • Exteins are now covalently joined

    • Structural Features

    Inteins are characterized by the HINT fold (Homologous to Inteins, N-terminal):

  • Horseshoe-shaped structure
  • Active sites at both termini
  • Highly conserved across all domains of life

    • Types of Inteins

    Cis-Splicing Inteins

  • Both exteins on the same polypeptide chain
  • Standard "contiguous" splicing

    • Trans-Splicing (Split) Inteins

  • Intein split into two pieces
  • Each piece fused to separate exteins
  • When they associate, splicing joins the exteins

    • - Enables protein trans-splicing

    Evolutionary Origins

    Inteins likely evolved as selfish genetic elements:

  • Many contain homing endonuclease domains
  • These promote intein gene propagation
  • Later evolved to minimal splicing elements
  • Found in all domains of life

    • Biotechnology Applications

    Inteins are powerful tools:

    Protein Purification

  • Intein-mediated purification (IMPACT system)
  • Self-cleaving affinity tags
  • No protease required

    • Protein Semi-Synthesis

    Expressed Protein Ligation (EPL):

  • Generate protein thioesters via intein
  • Ligate to synthetic peptides
  • Introduce non-natural amino acids, PTMs, labels

    • Segmental Labeling

  • Trans-splicing for NMR studies
  • Label only one segment for simplified spectra

    • Conditional Protein Activation

  • Inteins that splice in response to small molecules
  • Light-activated inteins
  • Protein activity switches
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    Short Linear Motifs (SLiMs)
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