title: Beta2-microglobulin can be refolded into a native state from ex vivo amyloid fibrils.
abstract: Beta2-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 beta2-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 beta2-microglobulin molecules and the main cleavage sites were at the C-terminus of Lys6 and Tyr10. Beta2-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. Beta2-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 beta2-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 beta2-microglobulin fragments that reduce the positive light polarisation at 205 nm typical of native beta2-microglobulin.
authors.affiliation: Department of Biochemistry, University of Pavia, Italy. stoppini@unipv.it
authors.forename: V
authors.initials: V
authors.lastname: Bellotti
authors.forename: M
authors.initials: M
authors.lastname: Stoppini
authors.forename: P
authors.initials: P
authors.lastname: Mangione
authors.forename: M
authors.initials: M
authors.lastname: Sunde
authors.forename: C
authors.initials: C
authors.lastname: Robinson
authors.forename: L
authors.initials: L
authors.lastname: Asti
authors.forename: D
authors.initials: D
authors.lastname: Brancaccio
authors.forename: G
authors.initials: G
authors.lastname: Ferri
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D001613:beta 2-Microglobulin