Stability and cooperativity in chimeric de novo TIM barrels via quarter swapping

The use of repetitive units as building blocks in protein design, mimicking evolutionary duplication events, is a valuable strategy that reduces structural complexity. Such modular designs offer an opportunity to dissect how local contributions are integrated in a context-dependent manner. sTIM11, the first validated de novo TIM barrel, and its derivatives, the DeNovoTIM collection, were designed following a four-fold repetition strategy. Here, we examine how chimeric constructs, generated by systematically swapping hydrophilic and hydrophobic quarters from two DeNovoTIMs, modify the energetic folding landscape. In-depth analysis of thermal unfolding transitions revealed a complex process under thermodynamic or kinetic control, as well as evidence of residual structure at high temperatures. Our findings show that swapping quarters stabilized by different types of interactions alters the thermodynamic stability of the barrel, favoring partially unfolded intermediates where stable quarters remain folded while less stable ones unfold, ultimately leading to a loss of cooperativity.