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

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."

Small and Random Peptides: An Unexplored Reservoir of Potentially Functional Primitive Organocatalysts. The Case of Seryl-Histidine

“…Fragment condensation of primitive peptides, unfortunately, lacks experimental studies, but it is per se worthy of more attention (note, however, that a protease-catalyzed oligomerization of a native protein segment in neat aqueous solution has been reported [ 100 ]). A qualitative account has been recently depicted by Luisi [ 101 ] and illustrated by means of an artificial procedure that led to one folded peptide (44 amino acids long) [ 102 ].…”

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.…”

Progress toward sourcing plants for new bioconjugation tools: a screening evaluation of a model peptide ligase using a synthetic precursor

“…The ability of AEPs to perform ligation has presented the opportunity to use them as a bioconjugation tool (Harris et al 2015;Nguyen et al 2016;Yang et al 2017). Other proteases that display ligase activity include sortase A and V8 protease from Staphylococcus aureus (Mazmanian et al 1999;Kumaran et al 2000), patG from cyanobacteria (Lee et al 2009), peptide cyclase 1 (PCY1) from Saponaria vaccaria of the family Caryophyllaceae (Barber et al 2013), and prolyl oligopeptidase B (POPB) from Galerina marginata (Luo et al 2014). In most of the aforementioned examples, the peptide ligases were discovered from efforts to identify the enzyme responsible for ligating cyclic peptides.…”