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 α-carboxyl and α-amino groups.
V8 protease catalyzed a chain of ligation steps between
pH 6 and 8 at 4 °C, 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.”