The rational design of proteins that maintain structural integrity under concurrent thermal, mechanical, and chemical stress remains a challenge in molecular engineering. We present a hierarchical framework that transforms an alpha-helical domain into an ultrastable scaffold by integrating AI-guided design with foundational chemical principles. This approach progresses from global architectural reinforcement, using multiple AI tools to create a stabilized four-helix bundle, to local chemical tuning, where AlphaFold3 guides the installation of salt bridges and metal-coordination motifs. A computational pipeline using physics-based screening such as molecular dynamics simulations efficiently distilled millions of designs into a minimal candidate set. The resulting alpha-helical proteins exhibit unprecedented multi-axis stability, with mechanical unfolding forces exceeding 200 pN, thermal resilience>100 degrees C, and high resistance to chemical denaturants. By systematically dissecting the contributions of hydrophobic packing, electrostatics, and metal coordination, we establish a general blueprint for imparting extreme robustness. This work bridges AI-driven structural generation with chemical precision, advancing the creation of durable proteins for mechanistic studies and synthetic biology.
[1]Nanjing Univ, Frontier Interdisciplinary Sci Res Ctr, Nanjing Drum Tower Hosp,Chem & Biomed Innovat Ctr, Sch Chem & Chem Engn,Affiliated Hosp,Med Sch, Nanjing, Peoples R China.
[2]Nanjing Univ, Nanjing, Peoples R China.
(8.0+极速模式)