Consensus-designed tetratricopeptide repeat proteins (CTPRs) are highly stable, modular proteins that are strikingly amenable to rational engineering. They therefore have tremendous potential as building blocks for biomaterials and biomedicine. Here we explore the possibility of extending the loops between repeats to enable further diversification, and we investigate how this modification affects stability and folding cooperativity. We find that extending a single loop by up to 25 residues does not disrupt the overall protein structure, but, strikingly, the effect on stability is highly context- dependent: In a two-repeat array, destabilisation is relatively small and can be accounted for purely in entropic terms, whereas extending a loop in the middle of a large array is much more costly, due to weakening of the interaction between the repeats. Our findings provide new insights into structure and folding that will be important both for understanding the function of natural repeat proteins and for the design of artifical repeat proteins in biotechnology.
