Variants of β₂-microglobulin cleaved at lysine-58 retain the main conformational features of the native protein but are more conformationally heterogeneous and unstable at physiological temperature

Cleavage of the small amyloidogenic protein β₂- microglobulin after lysine-58 renders it more prone to unfolding and aggregation. This is important for dialysis-related β₂- microglobulin amyloidosis, since elevated levels of cleaved β₂- microglobulin may be found in the circulation of dialysis patients. However, the solution structures of these cleaved β₂-microglobulin variants have not yet been assessed using single-residue techniques. We here use such methods to examine β₂-microglobulin cleaved after lysine-58 and the further processed variant (found in vivo) from which lysine-58 is removed. We find that the solution stability of both variants, especially of β₂-microglobulin from which lysine-58 is removed, is much reduced compared to wild-type β₂-microglobulin and is strongly dependent on temperature and protein concentration. ¹H-NMR spectroscopy and amide hydrogen (¹H/²H) exchange monitored by MS show that the overall three-dimensional structure of the variants is similar to that of wild-type β₂-microglobulin at subphysiological temperatures. However, deviations do occur, especially in the arrangement of the B, D and E β-strands close to the D-E loop cleavage site at lysine-58, and the experiments suggest conformational heterogeneity of the two variants. Two-dimensional NMR spectroscopy indicates that this heterogeneity involves an equilibrium between the native-like fold and at least one conformational intermediate resembling intermediates found in other structurally altered β₂-microglobulin molecules. This is the first single-residue resolution study of a specific β₂-microglobulin variant that has been found circulating in dialysis patients. The instability and conformational heterogeneity of this variant suggest its involvement in β₂-microglobulin amyloidogenicity in vivo.