Figures and data
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 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.
