Consensus tetratricopeptide repeat proteins are complex superhelical nanosprings

Tandem-repeat proteins comprise small secondary structure motifs that stack to form one-dimensional arrays with distinctive dynamic properties that are proposed to direct their cellular functions. Here, we report the force response of consensus-designed tetratricopeptide repeats (CTPRs) – superhelical arrays of short helix-turn-helix motifs. Not only are we able to directly observe the repeat-protein superhelix in the force data, but we also find that individual repeats undergo rapid fluctuations between folded and unfolded conformations resulting in a spring-like mechanical response. Using protein engineering, we show how the superhelical geometry can be altered by carefully placed amino-acid substitution and subsequently employ Ising model analysis to examine how these changes affect repeat stability and inter-repeat coupling in ensemble and single-molecule experiments. The Ising models furthermore allow us to map the unfolding pathway of CTPRs under mechanical load revealing how the repeat arrays are unzipped from both ends at the same time. Our findings provide the means to dissect and modulate repeat-protein dynamics and stability, thereby supporting ongoing research efforts into their functional relevance and exploiting them for nanotechnology and biomedical applications.

Intrinsic Disorder in Tetratricopeptide Repeat Proteins

Among the realm of repeat containing proteins that commonly serve as “scaffolds” promoting protein-protein interactions, there is a family of proteins containing between 2 and 20 tetratricopeptide repeats (TPRs), which are functional motifs consisting of 34 amino acids. The most distinguishing feature of TPR domains is their ability to stack continuously one upon the other, with these stacked repeats being able to affect interaction with binding partners either sequentially or in combination. It is known that many repeat-containing proteins are characterized by high levels of intrinsic disorder, and that many protein tandem repeats can be intrinsically disordered. Furthermore, it seems that TPR-containing proteins share many characteristics with hybrid proteins containing ordered domains and intrinsically disordered protein regions. However, there has not been a systematic analysis of the intrinsic disorder status of TPR proteins. To fill this gap, we analyzed 166 human TPR proteins to determine the degree to which proteins containing TPR motifs are affected by intrinsic disorder. Our analysis revealed that these proteins are characterized by different levels of intrinsic disorder and contain functional disordered regions that are utilized for protein-protein interactions and often serve as targets of various posttranslational modifications.