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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.