Context-Dependent Energetics of Loop Extensions in a Family of Tandem-Repeat Proteins

Albert Perez-Riba,Alan R Lowe,Ewan R.G Main,Laura S Itzhaki

doi:10.1016/j.bpj.2018.03.038

Abstract

Consensus-designed tetratricopeptide repeat proteins 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, destabilization 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 because of weakening of the interaction between the repeats. Our findings provide important and, to our knowledge, new insights that increase our understanding of the structure, folding, and function of natural repeat proteins and the design of artificial repeat proteins in biotechnology.

Highlights

Tandem-repeat arrays are one of the most common protein architectures
Comparison of the biophysical characteristics of all these different CTPR constructs enabled us to delineate the effects of loop insertion and of size of loop inserted versus the effects of point mutation
The 15 CTPR proteins consisted of the following: 1) a CTPR3 module, as studied previously by Regan and colleagues and referred to here as CTPR3-YD. This CTPR sequence contained a single-point mutation, Y91D, in the third repeat relative to other published CTPR sequences [41]; 2) a sixrepeat series built from two CTPR3-YD modules with either a native loop between the two modules (CTPR6-YD) or a poly-GS loop of differing length between 10 and 25 residues inserted between the two CTPR3-YD modules (CTPR6YD-loop10, CTPR6-YD-loop15, etc.)

Summary

Introduction

Tandem-repeat arrays are one of the most common protein architectures. The a-solenoids are one large family composed of such tandem-repeat arrays. Their repeats comprise between 12 and 45 amino acids that form pairs of antiparallel a-helices. Armadillo repeats, HEAT repeats (huntingtin, elongation factor 3, protein phosphatase 2A subunit, and the yeast kinase TOR1), and tetratricopeptide repeats (TPRs) [3,4,5,6]. They function in mediating protein-protein interactions by providing extended surfaces for molecular recognition. The modularity of their architectures has allowed the design of ultrastable consensus repeat proteins by selecting the most conserved residues in each family [7,8,9,10,11]

Methods

Results

Conclusion