The Effect of Loop Insertions on the Folding of Tandem-Repeat Proteins

Abstract

Multi-sequence alignment and the identification of conserved residues have allowed the design of highly stable consensus repeat proteins. The folding of repeat proteins has been shown to follow a 1-D Ising model, wherein fixed values for intra- and inter-repeat stability combined with the number of repeats are used to define the free energy of unfolding. Nevertheless, some questions remained unanswered, and we are using consensus-designed tetratricopeptide repeats (CTPRs) to address them: How are CTPR proteins able to remain soluble and stable without capping repeats? What role does the loop between adjacent repeats play in this folding paradigm? How do the loops contribute to inter-repeat stability? Here we explore the role of loops in the thermodynamics and kinetics of repeat protein folding by inserting variable-length unstructured loops at single or multiple sites along the repeat array. In particular we seek to understand how long loops are accommodated in the current folding models and to what extent the repeat protein is able to close these loops efficiently and still fold correctly. We also want to assess the feasibility of inserting functional peptide/protein motifs in between the stable inter-repeat interfaces so that we can expand the scope for exploiting repeat proteins as building blocks in biomaterials and the controlled geometric arrangement of function within them. Our preliminary results show that CTPR proteins with multiple loop insertions are stable and correctly folded. However, we find that stability no longer increases with the number of repeats, in contrast to what is observed in normal CTPR proteins and in striking deviation from the repeat protein folding paradigm. Further analysis is required to understand this breakdown in folding cooperativity and the implications for protein design.