Testing the length limit of loop grafting in a helical repeat protein

Alpha-helical repeat proteins such as consensus-designed tetratricopeptide repeats (CTPRs) are exceptionally stable molecules that are able to tolerate destabilizing sequence alterations and are therefore becoming increasingly valued as a modular platform for biotechnology and biotherapeutic applications. A simple approach to functionalize the CTPR scaffold that we are pioneering is the insertion of short linear motifs (SLiMs) into the loops between adjacent repeats. Here, we test the limits of the scaffold by inserting 17 highly diverse amino acid sequences of up to 58 amino acids in length into a two-repeat protein and examine the impact on protein folding, stability and solubility. The sequences include three SLiMs that bind oncoproteins and eleven naturally occurring linker sequences all predicted to be intrinsically disordered but with conformational preferences ranging from compact globules to expanded coils. We show that the loop-grafted proteins retain the native CTPR structure and are thermally stable with melting temperatures above 60 ​°C, despite the longest loop sequence being almost the same size as the CTPR scaffold itself (68 amino acids). Although the main determinant of the effect of stability was found to be loop length and was relatively insensitive to amino acid composition, the relationship between protein solubility and the loop sequences was more complex, with the presence of negatively charged amino acids enhancing the solubility. Our findings will help us to fully realize the potential of the repeat-protein scaffold, allowing a rational design approach to create artificial modular proteins with customized functional capabilities.