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.