Direct Structural Annotation of Membrane Protein Aggregation Loci using Peptide-Based Reverse Mapping

[Image: see text] Membrane protein aggregation is associated with neurodegenerative diseases. Despite remarkable advances to map protein aggregation, molecular elements that drive the structural transition from functional to amyloidogenic β-sheet polymers remain elusive. Here, we report a simple and reliable reverse-mapping method to identify the molecular elements. We validate our approach by obtaining molecular details of aggregation loci of human β-barrel nanopore ion channels that are vital for cell survival. By coupling bottom-up synthesis with time-resolved aggregation kinetics and high-resolution imaging, we identify molecular elements that switch folded channels to polymeric β-rich aggregates. We prove that intrinsic protein aggregation and amyloidogenicity does not depend on total hydrophobicity but on single residue differences in the primary sequence. Our method offers effective strategies for sequence-based design of aggregation inhibitors in biomedicine for neurodegenerative diseases.