Helix formation in enzymically ligated peptides as a driving force for the synthetic reaction: example of α-globin semisynthetic reaction

Rajendra Prasad Roy, K. M. Khandke, B. N. Manjula, A. S. Acharya

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11 Scopus citations

Abstract

The α-globin semisynthetic reaction, namely, the ligation of the complementary fragments of α-globin, α 1-30 and α 31-141, in the presence of 30% 1-propanol that is catalyzed by V8 protease is distinct as compared with the previously studied protease-catalyzed splicing of the discontinuity sites of the fragment complementing systems [Sahni et al. (1989) Biochemistry 28, 5456]. The complementary fragments of α-globin do not exhibit noncovalent interaction between them even in the presence of 1-propanol, the organic cosolvent used to facilitate the α-globin semisynthetic reaction. Besides, a significant portion of the fragment α 31-141 does not contribute to the protease-catalyzed splicing reaction. α 1-30 and α 31-40 are ligated by V8 protease to yield α 1-40 in much the same way as the splicing of α 1-30 with either α 31-141 or α 31-47 to yield α-globin or α 1-47, respectively. An equimolar mixture of α 1-30 and α 31-40 does not show any 'complexation' in the presence of 30% 1-propanol, the medium used for the synthetic reaction. The splicing junction, i.e., Glu 30-Arg 31 peptide bond, is located in the middle of the B-helix (residues 20-35) of the parent protein. Most of the residues from the A-helix of the protein could also be deleted from segment α 1-30 without influencing the V8 protease-catalyzed splicing reaction. V8 protease catalyzed the splicing of α 17-30 and α 31-40 in the presence of 30% 1-propanol with an overall yield of 40% that compares well with the yield of semisynthesis of full length α-globin. Segment α 17-40 exhibits very little α-helical conformation in the absence of the organic cosolvent. On the other hand, in the presence of 1-propanol, a significant amount of α-helical conformation is induced into α 17-40. However, the discontinuity at the Glu 30-Arg 31 peptide bond of α 17-40 completely abolished the α-helical conformation of the peptide even in the presence of the organic cosolvent. Therefore, we hypothesize that the increased helical conformation of the contiguous system (compared to the discontiguous system) in the presence of organic cosolvent operates as a 'conformational trap' of the semisynthetic reaction. Consistent with this hypothesis, V8 protease-catalyzed synthesis of α-globin and of α 1-47 from the respective complementary fragments proceeds smoothly in the presence of other helix-inducing organic cosolvents, like trifluoroethanol and 2-propanol. A corollary to this conformational trap hypothesis is that the splicing reaction will be independent of the protease used as far as the specificity requirements of the enzyme used are satisfied. Trypsin indeed catalyzed the ligation of Arg 31 of α 17-31 with Met 32 of α 32-40 in the presence of 1-propanol to generate the contiguous segment α 17-40. The results establish that the covalent contiguity generated by enzymic catalysis in a mixture of the complementary segments represents an assembly of a nascent segment with high 'α-helical conformational propensity'. The helical conformation is induced into the nascent contiguous peptide in the presence of the organic cosolvent, 1-propanol. This induction of α-helical conformation into the semisynthetic segment in the presence of the organic cosolvent generates the conformational trap, which acts as the driving force of the synthetic reaction. Thus, the α-globin semisynthetic reaction represents a new and novel class of protease-catalyzed protein/ peptide splicing reactions.

Original languageEnglish (US)
Pages (from-to)7249-7255
Number of pages7
JournalBiochemistry
Volume31
Issue number32
StatePublished - 1992
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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