<b>De novo design of dual-topology membrane transporters</b>

The transport of molecules across biological membranes is essential for life, allowing cells to acquire nutrients, remove waste, maintain cellular homeostasis and communicate with their environment. Although there have been advances in de novo design of functional transmembrane proteins, designing synthetic transporters that robustly and selectively transport specific small molecules across membranes has remained a significant challenge. In this study, we present the de novo design of dual-topology membrane transporters that achieve substrate-specific transport through a rationally programmed conformational cycle. By integrating symmetric backbone assembly with deep learning–guided sequence optimization, we designed 3-TM proteins that insert in opposite orientations and assemble into antiparallel dimers, forming a putative central substrate binding site that enables alternating access to either side of the membrane. These designed transporters mediate selective uptake of small-molecule dyes in both living cells and artificial liposomes, driven by substrate concentration gradients, resembling those of natural uniporters. Cryo-EM structures reveal high fidelity to the design models, and functional assays corroborate the dual-topology architecture and mechanism of action. Here we show that functional, dynamic membrane transporters can be built from the ground up with atomic-level precision—providing insights into the evolutionary origins of transporters and opening new avenues for applications, including targeted drug delivery and metabolic pathway engineering.