Figure 3. Electrophoretic examination of protein construct integrity as a function of exposure of Coh2-Flexible- BSX (3.4 μM) to Subtilisin A (varying concentrations annotated above the figure), using SDS-PAGE. The lane marked ‘C’ shows the Coh2-Flexible-BSX construct that was not exposed to Subtilisin A. The lane marked ‘MW’ shows molecular weight markers of 116, 66.2, 45, 35, 25, 18.4 and 14.4 kDa, respectively (top to bottom).
Understanding the counter-intuitive resistance to proteolysis of the ‘Flexible’ linker. An unstructured linker like Flexiblecould not possibly resist proteolysis without assistance from its flanking domains. Three kinds of assistance may be conceived to arise from steric hindrance offered by Coh2 and/or BSX to the approach of proteases, deriving from: (a) the sizes (or hydrodynamic volumes) of Coh2 and BSX; (b) the distance separating Coh2 and BSX; and (c) the motions of Coh2 and BSX, with respect to each other. Control experiments addressing the first two possibilities have already been described above, in that the sizes of the flanking domains are identical in all five constructs, and in that the physical proximity of Coh2 to BSX is also nearly identical in at least three constructs [Rigid (15 residues), Flexible (10 residues), and Nat-Quarter (9 residues)]; however, with proteolysis still seen to occur differentially in other constructs and the construct incorporatingFlexible . This establishes that it is neither the sizes (volumes) of Coh2 and BSX, nor the proximity of Coh2 and BSX, which are alone responsible for Flexible ’s remarkable resistance of proteolysis. This leaves only the third possibility, namely that differential susceptibilities arise from differences in the motions of Coh2 and BSX.Flexible presumably facilitates the greatest relative motions of its flanking domains, because it is flexible. It is not unreasonable to argue that such motions could serve to create large apparent hydrodynamic volumes of flanking domains (i.e., much bigger hydrodynamic volumes than actual volumes). This, in turn, could effectively create greater steric hindrance to the approach of proteases. In other words, the flanking domains probably function like ‘fans’ or ‘whisks’ that ‘bat-away’ any approaching proteases. Our work shows that an unstructured and flexible linker can arrange for its protection from proteases by imparting greater independence of motion to its flanking domains, and by receiving, in return, protection from proteolysis by approaching proteases.