Abstract
Repeat proteins are formed from units of 20–40 aa that stack together into quasi one-dimensional non-globular structures. This modular repetitive construction means that, unlike globular proteins, a repeat protein's equilibrium folding and thus thermodynamic stability can be analysed using linear Ising models. Typically, homozipper Ising models have been used. These treat the repeat protein as a series of identical interacting subunits (the repeated motifs) that couple together to form the folded protein. However, they cannot describe subunits of differing stabilities.Here we show that a more sophisticated heteropolymer Ising model can be constructed and fitted to two new helix deletion series of consensus tetratricopeptide repeat proteins (CTPRs). This analysis, showing an asymmetric spread of stability between helices within CTPR ensembles, coupled with the Ising model's predictive qualities was then used to guide reprogramming of the unfolding pathway of a variant CTPR protein. The designed behaviour was engineered by introducing destabilising mutations that increased the thermodynamic asymmetry within a CTPR ensemble. The asymmetry caused the terminal α-helix to thermodynamically uncouple from the rest of the protein and preferentially unfold. This produced a specific, highly populated stable intermediate with a putative dimerisation interface. As such it is the first step in designing repeat proteins with function regulated by a conformational switch.
Highlights
Repeat proteins are a diverse collection of superfamilies that are all composed of small modules (20– 40 aa), which are stacked together to form stable non-globular domains [1,2,3]
One elegant method used to dissect thermodynamic stability and folding cooperativity of both globular and repeat proteins is the fitting of their equilibrium denaturation curves or kinetics to Ising models
We and the Regan laboratory used a 1-D homozipper Ising model to analyse equilibrium denaturation data from consensus tetratricopeptide repeat proteins (CTPRs) protein series that differed in size by whole TPR motifs [9,16,33]
Summary
Repeat proteins are a diverse collection of superfamilies that are all composed of small modules (20– 40 aa), which are stacked together to form stable non-globular domains [1,2,3]. The stacking of the repeated units results in modular structures that are dominated by regularised interactions from residues close in primary sequence (both inter- and intra-repeat) These distinctive features result in a quasi one-dimensional (1-D) structure that has made repeat proteins extremely attractive folds to engineer, design and use as protein folding/stability models One elegant method used to dissect thermodynamic stability and folding cooperativity of both globular and repeat proteins is the fitting of their equilibrium denaturation curves or kinetics to Ising models. These statistical thermodynamic “nearest-neighbour” models have been used in many systems, both biological and non-biological, to describe order– disorder transitions [23]. Within the field of protein science, they have been used to probe helix to coil transitions, beta-hairpin formation, prediction of protein folding rates/thermodynamics and applied to interpret thermodynamic experiments that led to the postulation of downhill folding [23,24,25,26,27,28,29,30,31]
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