Flexible peptide linkers display higher resistance to proteolysis than
rigid linkers by facilitating movements that inhibit the approach of
proteases
Abstract
The differential proteolytic susceptibilities of different types of
linkers joining the same two protein domains were investigated, to
identify linkers displaying the highest resistance to degradation by
proteases in the environment. Five linkers, namely Rigid [3
repeats of EAAAK], Flexible [two repeats of SGGGG],
Nat-Full, Nat-Half and Nat-Quarter [3 linkers of
42, 21, and 9 residues, respectively, derived from a naturally-occurring
Coh2-adjacent sequence] were used to fuse two thermostable, and
proteolytically-resistant, domains, namely Coh2 (from C.
thermocellum CipA), and BSX (from Bacillus sp. NG-27). The
produced fusions were examined for linker degradation, using
electrophoretic (SDS-PAGE) and chromatographic (size exclusion)
experiments, following different periods of storage at 4 ⁰C.
Rigid, Nat-Full, Nat-Half and Nat-Quarter were degraded,
and released free Coh2 and BSX. However, Flexible displayed a
remarkable resistance to both environmental proteases and Subtilisin A
(a non-specific protease). Our data suggests that Flexible
imparts freedom of movement to Coh2 and BSX, and then reciprocally
receives protection against proteolysis from Coh2 and BSX, through their
dynamic steric inhibition (or ‘batting away’) of protease approach.