No abstract

We report an enigmatic peptide ligation reaction catalyzed by Glu-specific Staphylococcus aureus V8 protease that occurs in neat aqueous solution around neutral pH utilizing a totally unprotected peptide substrate containing free a-carboxyl and a-amino groups. V8 protease catalyzed a chain of ligation steps between pH 6 and 8 at 4 8C, producing a gamut of covalent oligomers~dimer through octamer or higher! of a native protein segment TAAAKFE~S39! derived from ribonuclease A~RNAse A!. Size-exclusion chromatography suggested the absence of strong interaction between the reacting peptides. The circular dichroism spectra of monomer through pentamer showed length-dependent enhancement of secondary structure in the oligomers, suggesting that protease-catalyzed ligation of a monomer to an oligomer resulted in a product that was more structured than its precursor. The relative conformational stability of the oligomers was reflected in their ability to resist proteolysis, indicating that the oligomerization reaction was facilitated as a consequence of the "conformational trapping" of the product. The ligation reaction proceeded in two phases-slow formation and accumulation of the dimer followed by a fast phase of oligomerization, implying that the conformational trap encountered in the oligomerization reaction was a two-step process. The Gly substitution at any position of the TAAAKFE sequence was deleterious, suggesting that the first step of the conformational trap, namely the dimerization reaction, that proceeded very slowly even with the parent peptide, was quite sensitive to amino acid sequence. In contrast, the oligomerization reaction of an Ala analog, AAAAKFE, occurred in much the same way as S39, albeit with faster rate, suggesting that Ala substitution stabilized the overall conformational trapping process. The results suggest the viability of the product-directed "conformational trap" as a mechanism to achieve peptide ligation of totally unprotected peptide fragments in neat aqueous solution. Further, the study projects the presence of considerable innate synthetic potential in V8 protease, baring rich possibilities of protein engineering of this enzyme to generate a "V8 peptide ligase."

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