Designed protein reveals structural determinants of extreme kinetic stability

Abstract

The design of stable, functional proteins is difficult. Improved design requires a deeper knowledge of the molecular basis for design outcomes and properties. We previously used a bioinformatics and energy function method to design a symmetric superfold protein composed of repeating structural elements with multivalent carbohydrate-binding function, called ThreeFoil. This and similar methods have produced a notably high yield of stable proteins. Using a battery of experimental and computational analyses we show that despite its small size and lack of disulfide bonds, ThreeFoil has remarkably high kinetic stability and its folding is specifically chaperoned by carbohydrate binding. It is also extremely stable against thermal and chemical denaturation and proteolytic degradation. We demonstrate that the kinetic stability can be predicted and modeled using absolute contact order (ACO) and long-range order (LRO), as well as coarse-grained simulations; the stability arises from a topology that includes many long-range contacts which create a large and highly cooperative energy barrier for unfolding and folding. Extensive data from proteomic screens and other experiments reveal that a high ACO/ LRO is a general feature of proteins with strong resistances to denaturation and degradation. These results provide tractable approaches for predicting resistance and designing proteins with sufficient topological complexity and long-range interactions to accommodate destabilizing functional features as well as withstand chemical and proteolytic challenge.SDS/protease resistance | protein folding | coarse-grained simulations | protein topology | contact order

Evidence for the principle of minimal frustration in the evolution of protein folding landscapes

“…The variation in the unfolding rate defines kinetic (unfolding rate) stability of proteins, and it was argued that one of the important consequences of the kinetic stability is to protect proteins from proteolytic degradation (59)(60)(61). To probe the correlation of the kinetic stability and resistance to proteolysis, we treated Trx samples with pepsin and thermolysin (62).…”

Quality over quantity: optimizing co-translational protein folding with non-‘optimal’ synonymous codons

“…The ability of these proteins to adopt distinct, stably folded structures via manipulation of their folding pathway indicates that these proteins fold under kinetic control, rather than to a single global energy minimum structure [31] (see Figure 3). Although proteins that fold under kinetic control are rarely used as models of protein refolding in vitro , a significant fraction of the proteome exhibits this behavior [32,33], which may be a designable feature [34]. …”

Detection of Folding Sites of β-Trefoil Fold Proteins Based on Amino Acid Sequence Analyses and Structure-Based Sequence Alignment

“…McLachlan [3] Murzin et al [4] Ponting et al [8] Lee et al [9,10] Broom et al [11,12] Longo et al [13,14] and Xia et al [15] pointed out that and proposed key residues in the three symmetrical units to form the β-Trefoil fold.…”