Transforming a Fragile Protein Helix into an Ultrastable Scaffold via a Hierarchical AI and Chemistry Framework

The rational design of proteins that maintain structural integrity under concurrent thermal, mechanical, and chemical stress remains a central challenge in molecular engineering. We present a hierarchical framework that transforms a fragile α-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 leveraging physics-based screening such as molecular dynamics (MD) simulations efficiently distilled millions of designs into a minimal candidate set. The resulting proteins exhibit unprecedented multi-axis stability, with mechanical unfolding forces exceeding 200 pN, thermal resilience >100 °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.