QUALITY OF THE HELIX INDUCED INTO ENZYMATICALLY LIGATED PEPTIDES DICTATES THE SPLICING OF NONINTERACTING PEPTIDES - A COMPARATIVE-STUDY OF THE V8 PROTEASE CATALYZED SPLICING OF COMPLEMENTARY SEGMENTS OF RNASE S-PEPTIDE AND ALPHA-GLOBIN

• Published in: Indian journal of chemistry. Sect. B. Organic chemistry, including medicinal chemistry Vol. 32; no. 1; pp. 1 - 10
• Main Authors: ACHARYA, AS, SAHNI, G, ROY, RP, MANJULA, BN
• Format: Journal Article
• Language: English
• Published: NEW DELHI Natl Inst Science Communication & Information Resources-Niscair 01.01.1993
• Subjects: Chemistry; Chemistry, Organic; Physical Sciences; Science & Technology
• ISSN: 0376-4699; 0019-5103

The alpha-globin semisynthetic reaction involving the V8 protease catalyzed splicing of alpha1-30 with alpha31-141, in the presence of n-propanol is distinct from the other protease mediated ligation of the ''discontinuity site'' of the fragment complementing systems [Sahni et al., Biochemistry, 28 (1989) 5456]. In this reaction, the ''complementation'' of the complementary fragments is not an essential molecular event for the splicing process. The alpha-helical conformation induced into the enzymically ligated contiguous segment by the organic cosolvent (n-propanol) has been suggested as the driving force for the alpha-globin semisynthetic reaction (Roy et al., Biochemistry, in press). Though V8 protease spliced the Glu30-Arg31 bond in an equimolar mixture of alpha1-30 and alpha31-141, or alpha1-30 and alpha31-47 or alpha17-30 and alpha31-40 in nearly the same yields, the chain contiguity mediated increase in the helical conformation in the three cases are distinct. Thus, the equilibrium yield of the splicing reaction does not correlate directly with the increase in the chain contiguity mediated n-propanol induced helical conformation of the contiguous peptide. In contrast to the facile splicing of alpha17-30 either with alpha31-47 or with alpha31-40 to generate the respective contiguous segments alpha17-47 or alpha17-40, the V8 protease did not ligate the complementary segments of RNase S-peptide (segments 1 to 9 and 10 to 20 of RNase A) in the presence of n-propanol, even though the formation of Glu9-Arg10 peptide bond is consistent with the specificity requirements of the enzyme and a significant level of alpha-helical conformation could be induced into the parent contiguous peptide (RNase S-peptide) in the presence of n-propanol. On the other hand, the formation of the Glu9-Arg10 peptide bond proceeded smoothly when RNase S-protein is incorporated into the system. V8 protease catalyzed splicing of the complementary segments of RNase S-peptide is not unique to the presence of n-propanol; it proceeded smoothly in the presence of 50% glycerol as well. The results demonstrate that the induction of an a helical conformation into an enzymically ligated nascent contiguous segment by itself does not serve as a universal ''conformational trap'' for facilitating the protease mediated splicing of non-interacting peptides. Apparently, the quality of n-propanol induced alpha-helical conformation of RNase S-peptide is distinct from that generated by the noncovalent interaction of the S-peptide with S-protein, the latter acting as the tertiary template for the induction of the halical conformation. Thus by analogy, it appears that the n-propanol induced alpha-helical conformation of alpha17-40 and alpha17-47 has integrated some unique structural features that mimic the role that RNase S-protein played in facilitating the semisynthesis of RNase S peptide from its complementary segments. A close examination of the amino acid sequence of alpha17-47 in this light suggested that an (i+4) side-chain interaction between the gamma-carboxyl of Glu-27 and the guanidino group of Arg-31 is possible in the alpha-helical conformation of the enzymically ligated contiguous segment. Engineering of this putative interaction by mutation of Glu-27 to Val resulted in nearly 50% loss of the splicing reaction. Therefore, contribution of (i+4) side-chain interaction appears to determine the quality of the propanol-induced helical conformation of alpha17-47 that facilitates the alpha-globin semisynthetic reaction. Delineation of unique conformational traps of the type discussed here is expected to provide a new dimension towards the design of novel strategies for the modular construction of proteins by chemical approaches.