Negative design enables cell-free expression and folding of designed transmembrane β-Barrels

De novo design of membrane proteins (MPs) is a rapidly growing field with transformative potential for synthetic biology. Yet, progress has lagged behind that of soluble proteins, largely due to limited understanding of the fundamental principles governing MP folding, stability, and solubility—and their integration into computational models. Here, we use a cell-free expression system to bypass the cytotoxicity of failed or insoluble designs and investigate how sequence features influence the folding of synthetic transmembrane β-barrels (TMBs). We find that even small, idealized TMBs challenge classical protein design workflows: sequences optimized solely for thermodynamic stability misfold and aggregate, preventing membrane insertion. Instead of bulk hydrophobicity, aggregation and β-sheet propensity emerge as key determinants of membrane association. By designing better folding variants of a synthetic TMB, we demonstrate that suppressing aggregation-prone intermediates through local destabilization of β-strands (“negative design”) significantly improves folding efficiency. Strikingly, even substitutions typically considered highly destabilizing, such as prolines or polar threonines exposed to the bilayer core, can improve folding when strategically positioned, without significantly compromising thermodynamic stability. Based on these findings, we propose a framework for joint optimization of native stability and folding pathways for future MP and nanopore design.