Significance Protein aggregation is a major problem for human health. However, our understanding of how folded proteins aggregate into amyloid lags behind. Using the tripartite β-lactamase assay (TPBLA) with our test protein, β 2 -microglobulin (β 2 m), we show the ability to differentiate the behavior of single-point variants and highlight the remarkable sensitivity to the identity of the residue at position 76. After evolving the aggregation-prone protein, D76N-β 2 m, the only mutations able to improve D76N-β 2 m behavior in vivo involve residues in a single 7-residue sequence of the protein. Further characterization in vitro shows that a single-point mutant in this region can abolish D76N-β 2 m aggregation.