The influence of Cu2+ on the unfolding and refolding of intact and proteolytically processed β2‐microglobulin

Human β2‐microglobulin (β2m) is an amyloidogenic protein in patients suffering from chronic kidney disease and especially in those patients that need intermittent hemodialysis for longer periods, e.g., when awaiting transplantation. While many in vitro conditions induce β2m‐amyloid formation from wild‐type (wt) β2m and while a number of structurally altered β2m molecules are known to be conformationally unstable and amyloidogenic on their own, it is not known why β2m‐amyloid is generated in some dialysis patients. For many amyloid proteins it is known that divalent metal ions, especially Cu2+, display strong binding and distinct destabilizing effects on protein conformation. The present study uses CE to assess conformational states of wt and cleaved β2m (dK58‐β2m, β2m cleaved at lysine‐58, a modification found in the circulation of hemodialysis patients) in the presence of divalent metal ions. The experiments provide both qualitative and quantitative data showing the specific destabilizing effects of Cu2+‐ions on the folding of wt β2m. Both refolding after acid denaturation and solution structure of β2m under otherwise native conditions are severely influenced by Cu2+. An increased unfolding, aggregation, and induction of Congo red‐reactive molecular species in Cu2+‐incubated wt‐β2m could be demonstrated while the refolding kinetics of dK58‐β2m, already slower than the wt molecule, appeared not to be further decreased by Cu2+. Given the interest in the actions of metal ions in other types of amyloidosis, including, e.g., Alzheimer's disease and the prion encephalopathies, the use of microelectrophoretic methods to monitor unfolding and refolding of biomolecules available in scarce amounts as shown in this study is an attractive option.