Post‐Translational Isoaspartate Promotes Amyloid Formation in β ₂ ‐Microglobulin

ABSTRACT β 2 ‐Microglobulin (β2m) aggregation is central to dialysis‐related amyloidosis (DRA), yet the molecular triggers underlying its fibrillogenesis remain incompletely defined. Among proposed mechanisms, isoaspartate (isoAsp) formation at Asn residues has been implicated but never directly tested due to synthetic inaccessibility. Here, we report the total chemical synthesis of β2m variants containing isoAsp at known in vivo hotspots, Asn 17 and Asn 42 , enabling precise structural and functional interrogation. Remarkably, the isoAsp 17 ‐modified β2m exhibited greater fibril formation capacity than the pathogenic ΔN6‐β2m variant, suggesting a previously underappreciated contribution of isoAsp 17 . Structural characterization was complemented by spectroscopic and TEM analyses, which demonstrated that isoAsp 17 promotes fibril formation. Our findings provide the first direct experimental evidence that a spontaneous post‐translational modification can initiate amyloidogenesis in β2m, redefining the molecular basis of DRA and highlighting isoAsp as a general driver of pathological protein aggregation.