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.

Keywords: SDS/protease resistance; coarse-grained simulations; contact order; protein folding; protein topology.

Fig. 1.

Design of ThreeFoil. (A) ThreeFoil (PDB: 3PG0) illustrating its three identical peptide subdomains (red, green, blue). (B) ThreeFoil’s secondary structure: turn (purple), β-strand/bridge (yellow), and 3/10-helix (magenta) and ligand binding residues indicated by colored circles and insertions shown in red. (C) Comparison of ThreeFoil with the independently designed Symfoil (PDB: 3O4D, 15% sequence identity), shown along (Left) and across (Right) the axis of symmetry. Backbones are colored by RMSD between the two structures (blue to white, 0–5 Å), with insertions in the loops of ThreeFoil relative to Symfoil colored red. ThreeFoil’s bound sodium shown in gray, and bis-Tris, which binds in the conserved carbohydrate binding sites, shown in cyan.

Fig. 2.

Folding and unfolding kinetics of ThreeFoil are modulated by ligand binding. (A) Chevron plots of observed folding and unfolding rate constants (in s⁻¹) in GuSCN were determined by fluorescence. Measurements were without sodium (gray open circles), with sodium (300 mM, black filled circles) or sodium and 50 mM of either lactose (cyan filled circles) or sucrose (cyan open circles). (B) Energy diagram corresponding to the kinetic measurements (coloring as in A). The energy axis is given by -RTln(k_obs) and the reaction coordinate follows the change in solvent accessible surface area as measured by m_f and m_u. The folded (F), transition (‡), and unfolded (U) states are indicated. Unfolded state energies and folded state reaction coordinates are set equal to facilitate comparisons.

Fig. 3.

ThreeFoil folding/unfolding kinetics are extremely slow compared with other proteins. Rate constants (gray diamonds) at the transition midpoint (ln(k_f) = ln(k_u)) for a large dataset of proteins (SI Appendix, Table S2) (41), are correlated with ACO (A) and LRO (B). β-trefoil proteins: ThreeFoil (orange), Symfoil (green), and Hisactophilin (blue) are highlighted. (C) The half-lives for folding (orange) and unfolding (blue) are shown for β-trefoils and the averages for the large dataset in each major structural class (α, β, αβ). The prototypical kinetically stable protein α-lytic protease is shown for comparison (35). Ankyrin proteins with 1–3 consensus designed internal repeats (NI1C to NI3C) illustrate the effect of increasing interface area and cooperativity (23).

Fig. 4.

Structure-based simulations reveal the molecular origins of ThreeFoil’s large kinetic barrier. (A) The folding free energy of ThreeFoil in units of k_BT_f (left y axis) is plotted at the transition midpoint (T_f) as a function of the fraction of native contacts (Q) in black. The population distribution is plotted in gray (right y axis). The protein populates only the unfolded state (Q ∼ 0.1) and the folded state (Q ∼ 0.9). The two curves were calculated from simulations of a ThreeFoil model using all contacts shown in D. (B) Contact map of the transition state ensemble (TSE, Q ∼ 0.35 in A), colored based on degree of structure, with 1 indicating native levels of structure and 0 indicating no structure. Contacts between lactose binding site residues (cyan) and sodium binding residues (gray) are shown. The numbered squares contain intratrefoil contacts (see C). (C) Average level of structure derived from B (same coloring) illustrated for ThreeFoil partitioned into its three repeats by gray lines. The residues shown as spheres are part of the 3 symmetric lactose-binding sites, whereas those shown as sticks are part of the single sodium-binding site (Fig. 1). The rotation indicated gives the view in E and F. (D) Contact map of ThreeFoil, with contacts deleted in MUT1 and MUT2 shown in red and blue, respectively. All deleted contacts are long-range (far from diagonal). (E) Long-range contacts (red sticks) of the β2–β3 loop residues (red spheres at Cα positions) deleted in MUT1. (F) For MUT2 the same number of contacts were deleted such that MUT1 and MUT2 have a very similar ACO. However, these contacts (blue sticks) are spread over the entire protein. (G) Folding free energies of ThreeFoil (black, same as in A), Symfoil (SymF; gray), a hybrid protein with the ThreeFoil contact map projected on the Symfoil backbone (HYB; gold), the two ThreeFoil mutants (MUT1; red, MUT2; blue), and Hisactophilin (His; green) are plotted in units of their respective folding temperatures (k_BT_f, y axis) at their respective transition midpoints as a function of the fraction of their respective native contacts (x axis). The SymF, HYB, MUT1 and MUT2 free energy profiles have very similar barrier heights, in between those of the highly kinetically stable ThreeFoil and the much less stable His.

Fig. 5.

ThreeFoil is highly resistant to protease and detergent. (A) Incubation with Proteinase K of: ThreeFoil (3F), Hisactophilin (His), human Cu,Zn superoxide dismutase (SOD), BSA, ovalbumin (Ova), β-lactoglobulin (βlac), myoglobin (Myo), and lysozyme (Lys). Protein before (−) and after incubation with protease (+) are shown. Retention of the protein band after incubation shows resistance to digestion. ThreeFoil and SOD are shown after 4 d (still nondegraded), whereas others are shown after 1 h (fully degraded). The molecular weight decrease for ThreeFoil after incubation is due to the loss of its unstructured his-tag (untagged ThreeFoil has a MW of 15.3 kDa and runs higher than intact Hisactophilin with a MW of 13.3 kDa, see also SI Appendix, Fig. S9A). Individual gels shown in SI Appendix, Fig. S9 B–E. (B) The same proteins tested for resistance to SDS. Where the unboiled (U) and boiled (B) samples are indistinguishable, no SDS resistance is observed, whereas a higher running unboiled sample indicates SDS is unable to penetrate and bind without thermal unfolding of the protein. Comparison of topological complexity as measured by ACO (C) and LRO (D) for proteins that have been kinetically characterized experimentally (SI Appendix, Table S2) and those with experimentally demonstrated resistance or nonresistance to protease and SDS (SI Appendix, Table S3). Resistant proteins generally have higher topological complexity. β-trefoil proteins are colored as in Fig. 3. Data shown as box-and-whisker plots, with a horizontal line at the median, box enclosing middle 50% of the data, whiskers drawn to 1.5*IQR (interquartile range).