Short flexible peptides linking non-interacting protein domains appear to resist proteolysis by facilitating domain motions that sterically inhibit protease approach

Abstract

Abstract Our objective in this paper was to identify any general characteristics of linker peptides that could potentially facilitate their escape from proteolytic degradation in fusion proteins, following their placement between two evolutionarily-unrelated, non-interacting, proteolytically-resistant protein domains. We selected Coh2 [an all-beta cohesin domain from C. thermocellum CipA], and BSX [a beta/alpha barrel xylanase domain from Bacillus sp. NG-27], and joined these domains through five different linker peptide sequences: (i) Rigid [3 repeats of N-EAAAK-C], (ii) Flexible [two repeats of N-SGGGG-C], (iii) Nat-Full [42 residues of a Coh2-adjacent linker in CipA], (iv) Nat-Half [a 21 residues-long derivative of Nat-Full] and (v) Nat-Quarter [a 9 residues-long derivative of Nat-Full]. The fusion proteins were produced, and size-exclusion chromatography and denaturing as well as non-denaturing electrophoresis were performed to assess the proteolytic susceptibilities of linkers peptide sequences during (a) storage of the fusion proteins at 4 ᵒC, through proteolysis effected by trace amounts of proteases naturally present in solution, and (b) exposure at room temperature to Subtilisin A, a non-sequence-specific protease. In both cases, proteolytic degradation of the linker was observed in Rigid, Nat-Full, Nat-Half and Nat-Quarter, but not in Flexible. Our further data and analyses suggest that, unlike with the other four other linkers, Flexible is able to escape proteolysis by using its conformational flexibility to facilitate motions in its flanking (non-interacting) domains to deflect approaching proteases, thus allowing such freely-moving domains to sterically protect the linker’s backbone, in ways that would be unlikely if the flanking domains were to interact.