Extreme stability in de novo-designed repeat arrays is determined by unusually stable short-range interactions

Abstract

Designed helical repeats (DHRs) are modular helix-loop-helix-loop protein structures that are tandemly repeated to form a superhelical array. Structures combining tandem DHRs demonstrate a wide range of molecular geometries, many of which are not observed in nature. Understanding cooperativity of DHR proteins provides insight into the molecular origins of Rosetta-based protein design hyperstability and facilitates comparison of energy distributions in artificial and naturally occurring protein folds. Here, we use a nearest-neighbor Ising model to quantify the intrinsic and interfacial free energies of four different DHRs. We measure the folding free energies of constructs with varying numbers of internal and terminal capping repeats for four different DHR folds, using guanidine-HCl and glycerol as destabilizing and solubilizing cosolvents. One-dimensional Ising analysis of these series reveals that, although interrepeat coupling energies are within the range seen for naturally occurring repeat proteins, the individual repeats of DHR proteins are intrinsically stable. This favorable intrinsic stability, which has not been observed for naturally occurring repeat proteins, adds to stabilizing interfaces, resulting in extraordinarily high stability. Stable repeats also impart a downhill shape to the energy landscape for DHR folding. These intrinsic stability differences suggest that part of the success of Rosetta-based design results from capturing favorable local interactions.

Functional instability allows access to DNA in longer transcription Activator-Like effector (TALE) arrays

“…By measuring the length-dependence of protein stability and employing a statistical mechanics Ising model, we previously described several different TALE partly folded states termed ‘end-frayed’, ‘internally unfolded’, and ‘interfacially fractured’ states. Although the calculated populations of partly folded states in TALE repeat arrays are small, they are many orders of magnitude larger than populations of partly folded states in other previously studied repeat arrays (consensus ankyrin [Aksel et al, 2011] and DHR proteins [Geiger-Schuller et al, 2018]) suggesting a potential functional role for the high populations of partially folded states in TALE repeat arrays.…”

Hierarchical design of multi-scale protein complexes by combinatorial assembly of oligomeric helical bundle and repeat protein building blocks

“…However, it can be seen that they deviate from the design model at the most distal portions of the structure. This is likely due to the inherent flexibility of the unsupported terminal helices of the DHRs 17,23,30 and lever arm effects which increase with increasing distance from the fusion site ( Figure S15).…”

Engineering repeat proteins of the immune system

“…Repeat domain architecture is characterized by tandem arrays of small structural motifs, typically 20 to 40 amino acids in length . Repeat domains are versatile, stable binding domains reliant on short‐range interactions for their stability . This architecture results in an extended structure with a large surface area and an elongated binding interface .…”