Product‐conformation‐driven ligation of peptides by V8 protease

Although the selective coupling of unprotected peptides through reverse proteolysis is one of the early protein semisynthetic reactions documented, delineation of molecular aspects that facilitate the reverse reactions of a protease continues to be a subject of considerable interest (Homandberg and Laskowski 1979; Wallace 1995; Nacharaju and Acharya 1999). Ligation of the discontinuity site of the fragment-complementing systems catalyzed by protease in the presence of organic cosolvent is the best studied example of this class of protein semisynthetic reaction (Homandberg and Laskowski 1979; Komoriya et al. 1980; Graf and Li 1981; Jullierat and Homandberg 1981; Homandberg et al. 1982; Iyer and Acharya 1987; Kullman 1987; De Filippis et al. 1990; Chang et al. 1994; Davey et al. 1995; Wallace 1995; Woods et al. 1996; Cerovsky et al. 1997; Nacharaju and Acharya 1999). In these systems, noncovalent interactions of two or more segments of a protein maintain a native-like folding despite the discontinuity in the polypeptide chain and provide the proximity to the ligating ends of the peptides, which is crucial for the protease-catalyzed splicing reaction. In the presence of organic cosolvent, the fragment complementation facilitates the protease-catalyzed splicing of the discontinuity site by reverse reaction. Accordingly, these protein-splicing reactions are referred to as conformationally assisted protein ligation reactions (Kullman 1987). Proteases have also been used to splice noninteracting peptides in the presence of organic cosolvents. Use of very high concentrations of one of the reactants (the amino compound) relative to the other, as well as trapping the product of the splicing reaction out of the equilibrium by using antibody (Nyberg 1988) or other proteins that strongly interact with the product, have been used as new strategies to achieve the reverse proteolytic reaction. The α-globin semisynthetic reaction, the V8 protease–catalyzed splicing of the segments (see Table 1​1 for the amino acid sequence of the peptides used in this study), namely, α1–30 and α31–141 of human HbA (reformation of E30-R31 peptide bond), is a new member of the class of protease-catalyzed splicing reactions of unprotected peptides (Nacharaju and Acharya 1999). However, this splicing reaction is unique and distinguishes itself from the previously described conformationally assisted splicing reactions in that it is the conformational aspects of the product that facilitates the splicing reaction (Sahni et al. 1989; Roy et al. 1992). Delineation of the molecular aspects of the product that facilitates the organic co-solvent-induced conformation of the product which in turn stabilizes the spliced peptide bond should pave the way for the translation of this reaction as a general approach to achieve the splicing of noninteracting peptides using protease. Table 1. The amino acid sequence of amino and carboxyl components of V8-catalyzed splicing reaction The thermodynamic stability of the conformational trap of the splicing reaction is sensitive to the chemical nature of the amino acid residues around the spliced junction, particularly to the presence or absence of the γ-carboxyl group of E27 and the guanidino group of R31 (Sahni et al. 1998). The segments α1–27 and α1–23 carry E at their carboxyl end and satisfy the specificity requirements of V8 protease; however, they do not undergo the splicing reaction with α31–40, α31–47, or α31–141 (Seetharam and Acharya 1986). Thus, the propensity of α17–30 to splice with α31–40 can be considered as the consequence of the ability of the sequence of α28–30 (i.e., ALE) to integrate the secondary structural information of α17–27 with that of α31–40. The γ-carboxyl group of E27 and the guanidino group of R31 play a significant role in establishing the maximum thermodynamic stability for the conformational trap of the splicing reaction (Sahni et al. 1998). Therefore the thermodynamic stability of E30-R31 peptide bond of α17–40 can be considered as the reflection of the integration of the α-helical potential of α17–26 with that of α32–40 by the pentapeptide sequence EALER to establish the product conformation. Accordingly, the primary structure of the peptides of α-globin for the purpose of stabilizing E30-R31 peptide bond against V8 protease can be generalized by the structure FRI-EALER-FRII (Fig. 1 ▶). Here, we refer to the pentapeptide EALER as the splicedon3 region that carries the information to splice the peptides, while the segments FR1and FRII are the flanking regions with some α-helical potential. In the V8 protease–catalyzed splicing reaction, the sequence EALE of the splicedon is at the carboxyl end of the FRI and R of the splicedon is at the amino terminal end of the FRII. Fig. 1. Schematic conceptualization of structural elements of the α-globin semisynthetic reaction. The complementary segments that generate the splicedon sequence EALER could be spliced. The pentapeptide EALER acts as the spacer group between two segments ... In this study, we investigate the influence of replacing the amino acid residues of splicedon with residues of lower α-helical propensity on the V8 protease resistance of the E30-R31 peptide bond. The influence of the exchanging of FRI and FRII region on the product conformational trap in the presence of 30% n-propanol, and its correlation with V8 protease resistance has also been established. A list of peptide designations and their amino acid sequence are shown in Table 1​1.. The results have led us to conceptualize that the thermodynamic stability of theV8 protease resistance of the E30-R31peptide bond in the contiguous segment reflect the potential of the splicedon region of the spliced peptides to flex between the helical (V8 protease resistant) and nonhelical (V8 protease susceptible) conformation. The rate of the helix-coil transition of the splicedon is apparently a correlation of its ability to integrate the α-helical propensities of the two flanking regions to lower the conformational transition around the spliced peptide bond relative to the rest of the molecule.