Integration of Evolutionary Analysis With RFdiffusion for De Novo Design of Aggregation-Resistant Frataxin
Proteins: Structure, Function and Bioinformatics, cilt.94, sa.6, ss.1225-1244, 2026 (SCI-Expanded, Scopus)
- Yayın Türü: Makale / Tam Makale
- Cilt numarası: 94 Sayı: 6
- Basım Tarihi: 2026
- Doi Numarası: 10.1002/prot.70114
- Dergi Adı: Proteins: Structure, Function and Bioinformatics
- Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, BIOSIS, Chemical Abstracts Core, EMBASE, INSPEC, MEDLINE
- Sayfa Sayıları: ss.1225-1244
- Anahtar Kelimeler: de novo design, frataxin, Friedreich's ataxia, molecular dynamics, protein aggregation, protein design, RFdiffusion
- Süleyman Demirel Üniversitesi Adresli: Evet
Özet
Friedreich's ataxia (FRDA) is a neurodegenerative disorder caused by frataxin (FXN) deficiency, where protein replacement therapy is hampered by the inherent instability and aggregation propensity of wild-type (WT) FXN. The structural flexibility of Loop-1 (residues 115–123), a critical region within the acidic ridge, represents a key determinant of protein stability. This study introduces a computational pipeline integrating evolutionary conservation analysis (ConSurf) with diffusion-based de novo design (RFdiffusion) to redesign both the backbone and sequence of Loop-1. Through systematic filtration of 1000 ProteinMPNN-generated variants using aggregation propensity screening (AGGRESCAN) and 450 ns of molecular dynamics (MD) simulations, four lead candidates were identified. Design_188 (EERVGGREI) demonstrated optimal performance with 2.3-fold improvement in aggregation resistance (Na4vSS: −53.8 vs. −23.5 for WT), superior structural stability (RMSD: 0.486 nm), reduced conformational diversity (62.3% dominant cluster occupancy), and 93% retention of ISCU binding capacity (ΔΔG: +6.4 kcal/mol). Experimental validation through 15N NMR relaxation analysis confirmed computational predictions, with Design_188 exhibiting uniform backbone rigidification (S2 = 0.81–0.95) and strong MD-NMR correlation (Pearson r = 0.675, p = 0.003). SEC-MALS analysis demonstrated near-complete monomeric behavior (> 98% monomer content) compared to WT's heterogeneous oligomerization (68% monomer, 32% oligomers), directly confirming the predicted anti-aggregation properties. K-means clustering analysis revealed an inverse relationship between conformational heterogeneity and stability, while correlation analysis identified a fundamental trade-off between aggregation resistance and structural stability (r = −0.82, p < 0.01). This work establishes a generalizable framework for therapeutic protein engineering where backbone redesign enables conformational ensemble modulation beyond the limitations of sequence optimization alone.