Product-conformation-driven ligation of peptides by V8 protease

Abstract

Organic co-solvent-induced secondary conformation of alpha(17-40) of human hemoglobin facilitates the splicing of E30-R31 in a mixture of its complementary segments by V8 protease. The amino acid sequence of alpha(17-40) has been conceptualized by the general structure FR(I)-EALER-FR(II) and the pentapeptide sequence EALER playing a major role in inducing the alpha-helical conformation. The primary structure of alpha(17-40) has been engineered in multiple ways to perturb one, two, or all three regions and the influence of the organic co-solvent-induced conformation and the concomitant resistance of E30-R31 peptide bond to V8 protease digestion has been investigated. The central pentapeptide (EALER), referred to here as splicedon,(3) appears to dictate a primary role in facilitating the splicing reaction. When the same flanking regions are used, (1) splicedons that carry amino acid residues of low alpha-helical potential, for example G at position 2 or 3 of the splicedon, generate a conformational trap of very low thermodynamic stability, giving an equilibrium yield of only 3%-5%; (2) splicedons with amino acid residues of good alpha-helical potential generate a conformational trap of medium thermodynamic stability and give an equilibrium yield of 20%-25%; (3) the splicedons with amino residues of good alpha-helical potential and also an amino acid that can generate an i, i + 4 side-chain carboxylate-guanidino (amino) interaction, a conformational trap of maximum thermodynamic stability is generated, giving an equilibrium yield of 45%-50%; and (4) the thermodynamic stability of the conformational trap of the spliced peptide is also influenced by the amino acid composition of the flanking regions. The V8 protease resistance of the spliced peptide bond is not a direct correlate of the amount of alpha-helical conformation induced into the product. The results of this study reflect the unique role of the splicedon in translating the organic co-solvent-induced product conformation as a site-specific stabilization of the spliced peptide bond. It is speculated that the splicedon with higher alpha-helical potential as compared to either one of the flanking regions achieves this by integrating its potential with that of the flanking region(s). Exchange of flanking regions with the products of other V8 protease-catalyzed splicing reactions will help to establish the general primary structural requirements of this class of splicing reactions and facilitate their application in modular construction of proteins.

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 with α-helical propensity (regions FR^I and FR^II) and the contiguous peptide assumes α-helical conformation in the presence of organic cosolvent and induces a degree of resistance to the V8 protease digestion of the E-R peptide bond of the contiguous peptide.

Fig. 2.

V8 protease–catalyzed splicing of α_17–30 and its truncated (either at its carboxyl end or internally) variants with α_31–40. RP-HPLC analytical pattern of an equimolar mixture of peptides after 48 h of reaction. The reaction mixture was analyzed on a protein C4 column (Vydac). The column was eluted with a linear gradient of 5%–50% acetonitrile containing 0.1% TFA in 100 min. The elution of the peptides was monitored at 210 nm. (A) Reaction mixture of α_17–30des_24–30 and α_31–40. No splicing reaction occurs in this incubation. The contiguous segment expected to be generated by the V8 protease–catalyzed splicing, namely α_17–40des_24–30, was chemically synthesized and its elution position is marked on the figure. (B) The reaction mixture of α_17–30des_23–26 and α_31–40. (C) The reaction mixture of α_17–30 and α_31–40. The inset compares the kinetics of the splicing of α_17–30 with α_31–40 (solid circles) with that of the splicing of α_17–30des_23–26 with α_31–40 (open circles). Both reactions reach an equilibrium in ∼24 to 30 h of the reaction.

Fig. 3.

Panel A shows the influence of the eight different splicedons on the equilibrium yields of the α_17–40des_23–26. Panel B shows the influence of changing the splicedon from EALER to EALEV on the equilibrium yields of the chimeric peptides. This influence is established by changing the amino-terminal R residue of α_31–40 and of β_30–39. In both cases, the respective complementary segments were incubated with V8 protease under conditions identical to that described in Fig. 2 ▶. and were analyzed by RP-HPLC after 48 h of incubation. Equilibrium yields were calculated from the integration of the peaks corresponding to the respective reactants and the products in the RP-HPLC maps.

Fig. 3.

Panel A shows the influence of the eight different splicedons on the equilibrium yields of the α_17–40des_23–26. Panel B shows the influence of changing the splicedon from EALER to EALEV on the equilibrium yields of the chimeric peptides. This influence is established by changing the amino-terminal R residue of α_31–40 and of β_30–39. In both cases, the respective complementary segments were incubated with V8 protease under conditions identical to that described in Fig. 2 ▶. and were analyzed by RP-HPLC after 48 h of incubation. Equilibrium yields were calculated from the integration of the peaks corresponding to the respective reactants and the products in the RP-HPLC maps.

Fig. 4.

Kinetics of the V8 protease digestion at the E-R peptide bond. The kinetics of digestion of various peptides was carried out as described in Materials and Methods. The concentration of the contiguous peptide was 1 mM during the start of the digestion. The inset shows the correlation of the equilibrium yields of splicing reactions of α_17–40 (solid squares), β_18–39(A²²,E²⁹) (open squares), α_17–40des_23–26 (open triangles), α_17–40des_23–26(Y²⁸,G²⁹) (open circles), and α_17–40des_23–26(G²⁸,A²⁹) (solid circles) with the rate of the digestion of these peptides.