Statistical thermodynamics based design principles into the temperature induced fold switching of a metamorphic protein

Fold-switching metamorphic protein sequences defy the classical “one sequence – one fold” paradigm. The ability of metamorphic proteins to reversibly switch between distinct folds make them attractive de novo protein engineering candidates since they can function as environment-sensitive molecular switches. However, the underlying thermodynamic design principles that drive their fold-switching behaviour is poorly understood. Gaining insights into the molecular driving forces leading to fold switching behavior is crucial for the rational design of new metamorphic proteins based molecular switches, sensors and stimulus responsive nanomaterials. In this study, we perform a detailed thermodynamic analysis of a designed fold-switching protein [ PNAS 2023, 120 (4), e2215418120] ¹ that transitions between a 3 α fold and α / β fold upon changes in temperature. We use an efficient advanced sampling molecular simulation based free energy calculation approach called Confine-Desolvate-Convert-Solvate-Release (CDCSR), which subjects the protein through the complete range of thermodynamic cycle and deconvolutes the enthalpic and entropic driving forces at each stage of the cycle. We find that while 3 α fold is stabilized at low temperatures by enthalpic contributions from favorable water-water and protein-water interactions, the transition to the α / β fold at high temperatures is driven by the gain of entropy from the release of ordered water molecules surrounding the 3 α fold. Our study elucidates the molecular driving forces governing temperature-induced fold-switching behavior and provides a rigorous statistical thermodynamic framework that can help in the design and engineering of future synthetic and functional metamorphic proteins.