CHEMISTRY OF THE MOLECULAR TRAP OF PROTEASE-CATALYZED SPLICING REACTION OF COMPLEMENTARY SEGMENTS OF ALPHA-SUBUNIT OF HEMOGLOBIN

The complementary fragments of human Hb alpha, alpha(1-30), and alpha( 31-141) are spliced together by V8 protease in the presence of 30% n-p ropanol to generate the full-length molecule (Hb alpha-semisynthetic r eaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb alpha is facilitated by the organic cosolvent induced alpha-helical conformation of product acting as the ''molecular trap'' of the splicing reaction. The segments alpha(24-30) and alpha(31-40) are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the con tiguous segment (product) alpha(24-40) has been manipulated by enginee ring the amino acid replacements to the positions alpha(27) and alpha( 31) to delineate the structural basis of the molecular trap. The locat ion of Glu(27) and Arg(31) residues in the contiguous segment alpha(24 -40) (as well as in other larger segments) is ideal to generate (i, i + 4) side-chain carboxylate-guanidino interaction in its alpha-helical conformation. The amino acid residue replacement studies have confirm ed that the side chains at alpha(27) and alpha(31) facilitate the semi synthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the s ide chain and the location. The gamma-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the therm odynamic 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 alpha-helical conformation to the contiguous segment in the presenc e of organic cosolvent that imparts some amount of protease resistance to Glu(30)-Arg(31) peptide bond. The second structural aspect is a si te-specific event, an i, i + 4 side-chain interaction in the alpha-hel ical conformation of the peptide which imparts an additional thermodyn amic stability to the molecular trap. The results suggest that conform ationally driven ''molecular traps'' of protease-mediated ligation rea ctions of peptides could be designed into products to facilitate the m odular assembly of peptides/proteins.