Conformationally driven protease‐catalyzed splicing of peptide segments: V8 protease‐mediated synthesis of fragments derived from thermolysin and ribonuclease A

Abstract

We have studied the conformation as well as V8 protease-mediated synthesis of peptide fragments, namely amino acid residues 295-316 (TC-peptide) of thermolysin and residues 1-20 (S-peptide) of ribonuclease A, to examine whether "conformational trapping" of the product can facilitate reverse proteolysis. The circular dichroism study showed cosolvent-mediated cooperative helix formation in TC-peptide with attainment of about 30-35% helicity in the presence of 40% 1 -propanol and 2-propanol solutions at pH 6 and 4 "C. The thermal melting profiles of TC-peptide in the above cosolvents were very similar. V8 protease catalyzed the synthesis of TC-peptide from a 1: 1 mixture of the noninteracting complementary fragments (TC295-302 and TC303-316) in the presence of the above cosolvents at pH 6 and 4 "C. In contrast, V8 protease did not catalyze the ligation of S1-9 and S10-20, although S-peptide could assume helical conformation in the presence of the cosolvent used for the semisynthetic reaction. V8 protease was able to synthesize an analog of S-peptide (SA-peptide) in which residues 10-14 were substituted (RQHMD + VAAAK). While S-peptide exhibited helical conformation in the presence of aqueous propanol solutions, SA-peptide displayed predominantly &sheet conformation. SA-peptide showed enhanced resistance to proteolysis as compared with S-peptide. Thus, failure of semisynthesis of S-peptide may be a consequence of high flexibility around the 9-10 peptide bond due to its proximity to the helix stop signal. The results suggest that protease-mediated ligations may be achieved by design and manipulation of the conformational aspects of the product.Keywords: conformation; organic cosolvent; proteosynthesis; reverse proteolysis; semisynthesis Recent years have seen remarkable progress in the area of synthetic protein chemistry toward developing strategies for chemical ligation of polypeptide fragments for construction of natural as well as novel proteins based on de novo design principles (Dawson & Kent, 1993;Dawson et al., 1994;Gaertner et al., 1994;Jackson et al., 1994;Liu & Tam, 1994;Tam et al., 1995;Wallace, 1995). The protease-catalyzed ligation of protein/peptide fragments offers the same flexibility to a selected segment as that available in modular chemical ligation strategy and may act as a bridge between chemical and genetic approaches for construction of pro--Reprint requests to: Rajendra P. Roy, National

An enigmatic peptide ligation reaction: Protease‐catalyzed oligomerization of a native protein segment in neat aqueous solution

“…The synthetic potential of V8 protease in organic cosolvents has been utilized in many studies concerning peptide ligation and semisynthesis of proteins that include synthesis of fragments of thermolysin and a-globin, a large domain of thermolysin containing noncoded Aib residues and hemoglobins containing chimeric a-globins~Sahni et al De Filippis & Fontana, 1990;Roy et al, 1993;Roy & Acharya, 1994;Kumaran et al, 1997;Nacharaju et al, 1997;De Filippis et al, 1998!. There also exists an example of a V8 protease-mediated transpeptidation reaction in aqueous buffer, albeit the fragments were held in close proximity by disulfide linkage~Canova-Davis et al, 1991!.…”

“…Furthermore, a protease-catalyzed reaction is also driven to synthesis with the use of a "molecular trap." In such cases, "trapping" of the product shifts the equilibrium to synthesis due to continuous removal of the spliced material from the chemical equilibrium Homandberg et al, 1982;Nyberg, 1988;Roy et al, 1992;Kumaran et al, 1997;Sahni et al, 1998!. The catalysis of amide bond formation by proteases proceeds as well in neat aqueous solution, but only with peptide esters as substrate. The systematic protein engineering of subtilisin by chemical modification, site-directed mutagenesis, and phage-display technology has yielded a variant enzyme, subtiligases, that can efficiently catalyze peptide ligation in aqueous solution~Nakatsuka et al, 1987;Wu & Hilvert, 1989;Abrahmsen et al, 1991;Chang et al, 1994;Braisted et al, 1997;Atwell & Wells, 1999!.…”

Shuffling peptides to create T‐cell epitopes: does the immune system play cards?

“…The precursor protein is cleaved and “re‐ligated” by an asparagine protease. “Reverse proteolysis” of several proteins and peptides has also been observed in vitro …”

“…"Reverse proteolysis" of several proteins and peptides has also been observed in vitro. [9][10][11][12] Recently, immunologists have begun to appreciate that reverse proteolysis can lead to the formation of T-cell epitopes. Our goal here is to review the discovery and the reports describing the analysis of T-cell epitopes formed by protease-mediated transpeptidation or protein splicing.…”