Exploring the folding energy landscape of a series of designed consensus tetratricopeptide repeat proteins

Abstract

Repeat proteins contain short, tandem arrays of simple structural motifs (20−40 aa). These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. One ubiquitous repeat is the tetratricopeptide motif (TPR), a 34-aa helix-turn-helix motif. In this article we describe the folding kinetics of a series of 7 designed TPR proteins that are assembled from arraying identical designed consensus repeats (CTPRa n ). These range from the smallest 2-repeat protein to a large 10-repeat protein (≈350 aa). In particular, we describe how the energy landscape changes with the addition of repeat units. The data reveal that although the CTPRa proteins have low local frustration, their highly symmetric, modular native structure is reflected in their multistate kinetics of unfolding and folding. Moreover, although the initial folding of all CTPRa n proteins involves a nucleus with similar solvent accessibility, their subsequent folding to the native structure depends directly on repeat number. This corresponds to an increasingly complex landscape that culminates in CTPRa10 populating a misfolded, off-pathway intermediate. These results extend our current understanding of the malleable folding pathways of repeat proteins and highlight the consequences of adding identical repeats to the energy landscape.

Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

“…Fluorescence and far-UV circular dichroism equilibrium unfolding measurements were performed as described previously. 17 Equilibrium data analysis. Data were analyzed in two specific ways.…”

“…By globally fitting a homozipper Ising model to chemical denaturations for a whole series of repeat proteins that differ only by their number of identical repeats, the intrinsic energy of a repeated unit and the interaction energy between the folded units can be delineated. 12,17,19,35 These values can then be summed to obtain the total stability of each protein within the series. We analyzed our denaturation data obtained at 25 C and compared it to previously published values under differing buffer conditions, but at the same temperature.…”

“…Recently, we and other groups have analyzed linear repeat protein equilibrium unfolding using a 1D homozipper Ising model. 12,17,19,34,35 This model treats each arrayed element of a repeat protein as an equivalent independently folding unit with nearest-neighbor pairwise interactions between those units. Thus, it breaks down the folding of a linear repeat protein into a linear series of interacting units.…”

“…1; the two series only differ by a 2-aa substitution per repeat). 11,12,17 NMR and equilibrium chemical denaturation studies on the CTPR2 and CTPR3 proteins suggest that, despite the apparent cooperative equilibrium unfolding, intermediate states with frayed terminal helices are populated through the denaturation transition. 23,24 Further, Regan and coworkers have used DSC to show that all the CTPRa proteins undergo multistate equilibrium folding under their conditions.…”

“…3,[9][10][11] Recently, the literature has shown great interest in the folding kinetics, cooperativity and equilibrium stability of repeat proteins. [12][13][14][15][16][17][18][19][20] In particular, studies have primarily focused on natural ankyrin repeat proteins and, to a lesser extent, TPR repeat proteins. These have investigated whether the modular nonglobular structures of repeat proteins inherently influence their folding and stability.…”