β2-Microglobulin can be refolded into a native state from ex vivo amyloid fibrils

Abstract

β2-microglobulin fibrils have been extracted from the femoral head of a patient who has been under chronic haemodialysis for 11 years. The primary structure of the N-terminal portion of the protein and mass determination by electrospray mass spectrometry demonstrate that β2-microglobulin, extracted as fibrils by the water extraction procedure, was not glycated and that Asn17 was not deamidated. Limited proteolysis was observed in more than 20% of β2- microglobulin molecules and the main cleavage sites were at the C-terminus of Lys6 and Tyr10. β2-microglobulin from fibrils has been purified by gel filtration in 6 M Gdn/HCl and submitted to a refolding procedure. The refolding conditions have been determined through the study of the unfolding pathway of the native protein. β2-microglobulin is stable at neutral pH where it displays a lower tendency to self-aggregate than in acidic conditions. Pulse dilution and extensive dialysis in refolding buffer at pH 7.5 yields β2-microglobulin with a tertiary structure identical to that of the native form. The CD spectrum in the near-ultraviolet region and the spectrum of the intrinsic fluorescence of Trp overlap those of the native protein, but the CD spectrum in the far-ultraviolet region is affected by the contribution of oligomers created by β2-microglobulin fragments that reduce the positive light polarisation at 205 nm typical of native β2- microglobulin.