Figure 2. Electrophoretic examination of protein construct
integrity as a function of storage, through monitoring by SDS-PAGE.Panel A. Behaviour of constructs on the day of purification.Panel B. Behaviour of constructs following 15 days of storage in
a refrigerator.
Counter-intuitive proteolytic susceptibilities of linker
peptides. What is most especially notable about Figure 2B is not the
observation that BSX and Coh2 are not vulnerable to degradation by
proteases present in the environment. This is expected, since they are
thermostable domains. Furthermore, prior work in our lab (data not
shown) had caused us to choose these domains for these experiments. What
is notable instead is the fact that the Coh2-Flexible- BSX
construct undergoes almost no degradation. This result is somewhat
counter-intuitive. The linker called Flexible cannot adopt a
particular structure, as it is constituted of two serine residues and
eight glycine residues (since glycine possesses only a hydrogen atom as
its side chain, and this typically imparts great conformational freedom
to regions of proteins chains that contain the residue, glycine).Flexible is, in fact, a popular linker used in protein fusions
(Argos, 1990; Chen et al ., 2013) precisely because its own lack
of intrinsic structure prevents it from restricting the motions (and
activities) of flanking domains. Our results suggest thatFlexible undergoes no proteolysis despite remaining unstructured,
although it is the least likely of five different linkers to undergo
proteolysis, when placed between well-folded domains that themselves
resist proteolysis. It must be remarked that, in experiments involving
poorly-folded flanking domains, proteolysis can (and does) occur at
multiple sites upon storage, with this ordinarily smearing-out any
distinctions between ‘proteolytic vulnerabilities of linkers’ and
‘proteolytic vulnerabilities of flanking domains’. Here, having ensured
that no proteolysis occurs in flanking domains, we observe that the
linker least likely to resist proteolysis is actually the one that
maximally resists proteolysis. Furthermore, this result is obtained
after ensuring that every parameter remains equal in all conducted
experiments, i.e., with linkers possessing identical flanking domains
(with identical conformational stabilities), and with fusions subjected
to identical physical and chemical conditions for testing, and identical
methods of testing, using identical protein concentrations, and
identical durations of incubation.
Probing of the proteolytic susceptibility of the ‘Flexible’ linker
through use of Subtilisin A. Before proceeding to rationalize the
counter-intuitive result that an unstructured linker peptide such asFlexible undergoes the least proteolysis, we decided to further
examine the resistance of Flexible to proteolysis in the context
of its presence in Coh2-Flexible- BSX, by exposing
Coh2-Flexible- BSX to the non-specifically-acting serine protease,
Subtilisin A, using 2 h of incubation and different molar ratios of
Subtilisin A:Coh2-Flexible -BSX. Subtilisin A is commonly used to
test the relative proteolytic vulnerabilities of different regions of a
folded protein, as well as to probe for regions that are not as
well-folded as other regions, through limited proteolytic digestion
performed using varying Subtilisin A:protein ratios, and/or varying
durations of incubation (Sharma and Guptasarma, 2008). In such
experiments, Subtilisin A helps to identify regions that are
poorly-structured in a significant fraction of the population at any
given time, since enough time is not given for the protease to degrade
the entire population into small peptides, the assumption being that the
nature of the folding equilibrium determines both (a) the initial
species formed through limited proteolytic digestion prior to
complete digestion, and (b) the time required for complete degradation
(or limit digestion ).
Ordinarily, Subtilisin A:protein ratios of 1:1000 (or 0.001:1) degrade
unstructured regions of proteins within minutes (or tens of minutes) of
exposure. Figure 3 shows that virtually the entire population of
Coh2-Flexible -BSX remains un-degraded even after 2 h of
incubation with Subtilisin A, using a Subtilisin
A:Coh2-Flexible -BSX ratio of 0.001:1, with only hints of
separated BSX and Coh2 bands being visible. At ratios that are more
favourable to proteolysis, substantial fractions of the population can
still be seen to remain un-degraded, and significant degradation is seen
only when relative concentrations of Subtilisin A and
Coh2-Flexible -BSX approach the same order of magnitude. Only when
a ratio of 1:1 is used is Coh2-Flexible -BSX observed to have been
completely destroyed. As the relative amounts of Subtilisin A rise to
high levels, and before complete degradation is observed, some
degradation can be seen to have occurred even in the BSX and Coh2
domains, with this generating some additional gel bands (left unmarked
in Figure 3).