Epistasis on the stability landscape of de novo TIM barrels explored by a modular design approach
The ability to design stable proteins with custom-made functions is a major goal in biochemistry with practical relevance for our environment and society. High conformational stability lowers protein sensitivity to mutations and changes in the environment; thus, understanding and manipulating protein stability will expand the applications of de novo proteins. Since the ({beta}/)8-barrel or TIM-barrel fold is one of the most common functional scaffolds, in this work we designed a collection of stable de novo TIM barrels (DeNovoTIMs), using a computational fixed-backbone and modular approach based on improved hydrophobic packing of sTIM11, the first validated de novo TIM barrel. DeNovoTIMs navigate a region of the stability landscape previously uncharted by natural TIM barrels, with variations spanning 60 degrees in melting temperature and 22 kcal per mol in conformational stability throughout the designs. Significant non-additive or epistatic effects were observed when stabilizing mutations from different regions of the barrel were combined. The molecular basis of epistasis in DeNovoTIMs appears to be related to the extension of the hydrophobic cores. This study is an important step towards the fine-tuned modulation of protein stability by design. Significance StatementDe novo protein design expands our knowledge about protein structure and stability. The TIM barrel is a highly relevant fold used in nature to host a rich variety of catalytic functions. Here, we follow a modular approach to design and characterize a collection of de novo TIM barrels and subjected them to a thorough folding analysis. Non-additive effects modulate the increase in stability when different regions of the barrel are mutated, showing a wide variety of thermodynamic properties that allow them to navigate an unexplored region of the stability landscape found in natural TIM barrels. The design of stable proteins increases the applicability of de novo proteins and provides crucial information on the molecular determinants that modulate structure and stability. One Sentence SummaryA family of designed TIM barrels with diverse thermodynamic properties shows epistatic effects on its stability landscape.