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).