Distinct energetic blueprints diversify function of conserved protein folds

Advances in structural biology have improved our understanding of the relationship between protein structure and function, while also confirming a widely applicable principle: protein domains with highly conserved three-dimensional folds can perform radically disparate biochemical functions. To gain insight into this structural enigma, we mapped the energetic landscapes of a family of bacterial transcription factors and their anciently diverged structural homologues, the periplasmic binding proteins. Using hydrogen exchange-mass spectrometry, bioinformatics, X-ray crystallography and molecular dynamics, we uncovered an unexpected contrast: despite binding the same sugars, the two families have evolved unique 'energetic blueprints' to support their distinct functional requirements. To test if differences in ensemble energies have functional consequences, we rationally redesigned the protein fold for tunable ligand-driven transcriptional responses. Strikingly, energy-driven protein engineering produced synthetic transcription factors with the theoretically anticipated ligand-induced transcriptional outputs. Thus, decoding energetic blueprints among conserved protein folds provides diverse functional adaptations, paves an alternative roadmap for protein design, and offers a distinct approach for engineering challenging drug targets.