Chemistry of the 'molecular trap' of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

Girish Sahni, Shabbir A. Khan, A. Seetharama Acharya

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6 Scopus citations


The complementary fragments of human Hb α, α1-30 and α31- 141 are spliced together by V8 protease in the presence of 30% n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the 'molecular trap' of the splicing reaction. The segments α24-30 and α31-40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α24-40 has been manipulated by engineering the amino acid replacements to the positions α27 and α31 to delineate the structural basis of the molecular trap. The location of Glu27 and Arg31 residues in the contiguous segment α24-40 (as well as in other larger segments) is ideal to generate (i, i + 4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α27 and α31 facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu30-Arg31 peptide bond. The second structural aspect is a site- specific event, an i, i + 4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven 'molecular traps' of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.

Original languageEnglish (US)
Pages (from-to)669-678
Number of pages10
JournalJournal of Protein Chemistry
Issue number7
StatePublished - Oct 1998


  • I, i + 4 side- chain interaction
  • Protein/peptide folding
  • Reverse proteolysis
  • α-helix

ASJC Scopus subject areas

  • Biochemistry


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