Abstract
Ninety-five percent of all transmembrane proteins exist in kinetically trapped aggregation-prone states that have been directly linked to neurodegenerative diseases. Interestingly, the primary sequence almost invariably avoids off-pathway aggregate formation, by folding reliably into its native, thermodynamically stabilized structure. However, with the rising incidence of protein aggregation diseases, it is now important to understand the underlying mechanism(s) of membrane protein aggregation. Micromolecular physicochemical and biochemical alterations in the primary sequence that trigger the formation of macromolecular cross-β aggregates can be measured only through combinatorial spectroscopic experiments. Here, we developed spectroscopic thermal perturbation with 117 experimental variables to assess how subtle protein sequence variations drive the molecular transition of the folded protein to oligomeric aggregates. Using the Yersinia pestis outer transmembrane β-barrel Ail as a model, we delineated how a single-residue substitution that alters the membrane-anchoring ability of Ail significantly contributes to the kinetic component of Ail stability. We additionally observed a stabilizing role for interface aliphatics, and that interface aromatics physicochemically contribute to Ail self-assembly and aggregation. Moreover, our method identified the formation of structured oligomeric intermediates during Ail aggregation. We show that the self-aggregation tendency of Ail is offset by the evolution of a thermodynamically compromised primary sequence that balances folding, stability, and oligomerization. Our approach provides critical information on how subtle changes in protein primary sequence trigger cross-β fibril formation, with insights that have direct implications for deducing the molecular progression of neurodegeneration and amyloidogenesis in humans.