Decoupling a tandem-repeat protein: Impact of multiple loop insertions on a modular scaffold

Abstract

The simple topology and modular architecture of tandem-repeat proteins such as tetratricopeptide repeats (TPRs) and ankyrin repeats makes them straightforward to dissect and redesign. Repeat-protein stability can be manipulated in a predictable way using site-specific mutations. Here we explore a different type of modification - loop insertion - that will enable a simple route to functionalisation of this versatile scaffold. We previously showed that a single loop insertion has a dramatically different effect on stability depending on its location in the repeat array. Here we dissect this effect by a combination of multiple and alternated loop insertions to understand the origins of the context-dependent loss in stability. We find that the scaffold is remarkably robust in that its overall structure is maintained. However, adjacent repeats are now only weakly coupled, and consequently the increase in solvent protection, and thus stability, with increasing repeat number that defines the tandem-repeat protein class is lost. Our results also provide us with a rulebook with which we can apply these principles to the design of artificial repeat proteins with precisely tuned folding landscapes and functional capabilities, thereby paving the way for their exploitation as a versatile and truly modular platform in synthetic biology.

Consensus tetratricopeptide repeat proteins are complex superhelical nanosprings

“…S11). This directionality is a natural consequence of the array geometry itself, since repeats at the centre of the array are less likely to unfold than those at the termini, but it is also consistent with hydrogen-deuterium exchange experiments of CTPRs [27,64] and further studies of other designed and natural repeat proteins. For example, consensus ankyrin repeats were shown to unfold from both ends using chemical denaturation [58] and from one end to the other under force [39].…”

“…As such, our findings on the unfolding pathway are consistent with the literature showing that unfolding of various consensus repeat arrays occurs from either end, albeit with different probabilities. They are furthermore supported by the fact that the terminal repeats of CTPRs are subject to higher hydrogen-deuterium exchange rates [36, 53], and hence they are more likely to explore the unfolded state than more central repeats. Our Ising models even confirm the folding correlation length of 3 repeats and the possibility of multiple folding “seeds” that were previously determined based on coarse-grained simulations [42].…”

“…The functions of TPR proteins are diverse, ranging from scaffolds of multi-protein assemblies and regulators of cell division to molecular chaperones and mediators of bacterial quorum sensing [21,[23][24][25]. Consensus-designed TPRs (CTPRs) are good candidates for building 'made to measure' proteins, because they form stable arrays and are very amenable to manipulation including loop insertions [26][27][28]. Their chemical stability has been characterised previously [22,27,[29][30][31][32][33][34][35][36], whereas, in contrast to other repeat proteins [12,14,[37][38][39][40][41], their mechanical properties remain unexplored.…”

“…Consensus-designed TPRs (CTPRs) are good candidates for building 'made to measure' proteins, because they form stable arrays and are very amenable to manipulation including loop insertions [26][27][28]. Their chemical stability has been characterised previously [22,27,[29][30][31][32][33][34][35][36], whereas, in contrast to other repeat proteins [12,14,[37][38][39][40][41], their mechanical properties remain unexplored. For the above reasons, we chose TPRs to examine how repeat energetics are connected to both the shape and the mechanics of the superhelix.…”

“…Both natural and consensus TPR structures have been the subject of intensive study, because their simple structures make them strikingly amenable to both the dissection and the redesign of their biophysical properties, with tremendous potential for exploitation in biotechnology and medicine [26,27]. Their chemical stability has been described in depth [3,[28][29][30][31][32][33][34][35][36], whereas their mechanical properties remain unexplored even though they are one of the repeat protein types whose structures most closely resemble physical springs.…”