Beta2-microglobulin can be refolded into a native state from ex vivo amyloid fibrils.
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.
Bellotti
V
V
Department of Biochemistry, University of Pavia, Italy. stoppini@unipv.it
Stoppini
M
M
Mangione
P
P
Sunde
M
M
Robinson
C
C
Asti
L
L
Brancaccio
D
D
Ferri
G
G
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
D001613:beta 2-Microglobulin