A hierarchical chemical blueprint for multi-axis stabilization of α-helical protein.

a, The erythrocyte membrane skeleton, which relies on spectrin repeats to withstand shear stress. Inset: Structure of a single, naturally fragile spectrin repeat (PDB 3F57), with hydrophobic core residues shown. b, The mechanical unfolding pathway of a spectrin repeat under tensile force, probed computationally by steered molecular dynamics (SMD) to generate force-extension curves. c, Overview of the two-stage design strategy. Stage I (Architectural Stabilization): AI-guided backbone construction and computational screening generate four-helix designs with optimized hydrophobic cores. Stage II (Precision Functionalization): Rational installation of inter-helical salt bridges and metal-coordination motifs reinforces specific mechanical interfaces. d, Stage I, Backbone construction. RFdiffusion appends a fourth helix to the native three-helix template, generating 100 initial backbones. Five optimal four-helix scaffolds are selected, and ProteinMPNN is used to generate 100,000 sequences per scaffold. e, Stage I, Computational screening. A multi-step funnel prioritizes candidates through successive filters: developability (GRAVY score ≤ −0.3), foldability (ESMFold triage followed by AlphaFold2 refinement with RMSD ≤ 2.0 Å and pLDDT ≥ 90), and stability assessed via molecular dynamics. The process efficiently narrows ∼106 initial designs to an experimentally tractable shortlist.

Stability of AI-designed spectrin with four α-helices

Thermal and chemical robustness of AI-designed spectrin variants.

a, MALDI-TOF mass spectrometry confirm molecular weights for SpecAI88, SpecAI41, and SpecAI89. b, Far-UV circular dichroism (190–260 nm) shows α-helical signatures with minima at 208 and 222 nm. Temperature-dependent CD (20–100 °C, blue to red) indicates substantial retention of ellipticity at 195 nm, with melting temperatures exceeding 100 °C. c, CD at 222 nm recorded in guanidinium chloride (GdnHCl) demonstrates persistence of α-helical signal at high denaturant concentrations (∼3 M), indicating high chemical resistance.

Stage I four-helix bundle designs exhibit enhanced mechanical stability by single-molecule force spectroscopy.

a, Schematic of the AFM–SMFS experimental setup. A dockerin (Doc)-functionalized AFM tip engages a cohesin (Coh)-tagged SpecAI construct immobilized on the surface, enabling single-molecule pulling. The construct includes three GB1 domains as fingerprint markers. b, Representative force-extension curves. Curve 1: Unfolding of the natural spectrin repeat (ΔLc ≈ 32 nm, red), followed by three GB1 (ΔLc ≈ 18 nm per domain, black). Curves 2-4: Unfolding of SpecAI variants (ΔLc ≈ 53 nm). Sometimes, the Doc unfolds showing additional peak (Curve 3). Dashed lines are worm-like chain model fit. c, Unfolding force histograms of SpecAI (bin size= 30 pN) with Gaussian fits demonstrate a significant increase in mechanical stability compared to the native spectrin repeat (56 ± 3 pN, n = 212, bin size=13.75 pN): SpecAI88, 116 ± 2 pN (n = 224); SpecAI41, 156 ± 4 pN (n = 218) and SpecAI89, 121 ± 4 pN (n = 174). The corresponding ΔLc distributions are centered near 52 nm, consistent with the full unfolding of the designed domain.

Precision stabilization of designed proteins via electrostatic interactions and metal coordination.

a Schematic of Stage II design: introducing inter-helical ion pairs and metal-coordination sites into AI-designed backbones to stabilize specific interfaces. b, AlphaFold3-predicted structure of variant SpecAI41-9K152D, showing an engineered salt bridge (Lys9-Asp152, 3 Å) designed for electrostatic stabilization without perturbing the core. The color of protein is based on the pLDDT value, showing a high confidence structure prediction (>80). c, A representative force-extension curve for the salt-bridge variant shows an unfolding event with a ΔLc of 53 nm, consistent with the parent scaffold. d, Unfolding force histograms reveal a ∼25 pN increase in mechanical stability for salt-bridge variants compared to their Stage-I parents. e, Introduction of a metal-binding site in variant SpecAI41-9K152D-6H153H, with two histidines positioned 2 Å apart, compatible with Ni2+ coordination. f, A representative force-extension curve recorded in 200 µM Ni2+ shows the unfolding event (ΔLc ≈ 53 nm). g, Unfolding force histograms confirm enhanced mechanical stability, with forces reaching ∼200 pN, a significant gain over the salt-bridge parent.

SpecAI stability further enhanced by site-specific functionalization

Hierarchical computational design of ultrastable proteins through multi-scale stabilization.

Schematic summarizing the two-stage design strategy for additive mechanical reinforcement. Stage I establishes a stable architectural framework through MD screening of AI-designed hydrophobically optimized cores. Stage II introduces precision functionalization via inter-helical salt bridges and bi-histidine metal-coordination motifs, guided by AlphaFold3 structural models. The integration of global architectural stability with local chemical cross-linking produces additive mechanical reinforcement, enabling the creation of ultrastable protein domains.