Flexible peptide linkers display higher resistance to proteolysis than rigid linkers by facilitating movements that inhibit the approach of proteases

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.