The solution structure and stability of N-terminally truncated b2-microglobulin~DN6b2-m!, the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that DN6b2-m has a free energy of stabilization that is reduced by 2.5 kcal0mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full-length proteins at mM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that DN6b2-m is significantly less protected than its wild-type counterpart. Analysis of DN6b2-m by NMR shows that this loss of protection occurs in b strands I, III, and part of II. At mM concentration gel filtration analysis shows that DN6b2-m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of b2-microglobulin was found to have a higher tendency to self-associate than the intact molecule, and unlike wild-type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that DN6b2-m could be a key intermediate of a proteolytic pathway of b2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of b2-microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between b-strands far removed from this constrain is greatly perturbed.Keywords: amyloidosis; b2-microglobulin; hydrogen exchange mass spectrometry; limited proteolysis; NMR; protein folding Amyloidoses are diseases caused by tissue deposition of protein aggregate organized in an ordered b-sheet structure. The conversion of globular proteins to insoluble fibrillar aggregates requires significant conformational changes, such as the loss of tertiary and quaternary interactions or conversion of a to b secondary structurẽ Sunde & Blake, 1998!. Of the 17 or so proteins implicated in amyloidoses the fibril morphology is indistinguishable and there does not appear to be any common features that link the soluble precursor proteins. For many of these proteins, the amyloid fibril formation is facilitated by amino acid mutations that destabilize the native state and confer a structural flexibility to the molecule, but other proteins like IAPP, wild-type TTR, and b2-microglobulin

Ϫ EJB 98 0390/3 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.Keywords : amyloidosis; fibril; β2-microglobulin; structure ; refolding.Chronic kidney failure and the haemodialytic procedure are associated with a high prevalence of a particular form of amyloidosis, mainly involving the muscle-skeletal system, in which the amyloid fibrils are composed of β2-microglobulin (β2-m) [1]. In this form, as in all of the amyloidoses, the fibrillar deposits represent the central component of the disease; this component exists in a dynamic equilibrium between soluble amyloidogenic protein precursors on the one hand and the amyloid catabolic pathway on the other. The progression of the disease requires that protein accumulation is favoured, but in certain cases amyloid deposits can be partially or completely reabsorbed when the supply of amyloidogenic precursor is reduced [2] as in AA and AL amyloidosis or in some cases of dialysis-related amyloidosis (DRA) after kidney transplantation. The question of β2-m reabsorption in DRA patients submitted to kidney transplantation is still open. The group of Pepys has shown by SAP scan, a reduction of the amyloid deposits [3], but other groups have bioptically demonstrated the persistence of amyloid fibrils in the deposits ten years after the renal transplantation [4]. The mechanism of amyloid reabsorption in this form, as in the other types of amyloidosis, is not known, but it can probably follow Correspondence to V. Bellotti, Dipartimento di Biochimica, Università degli Studi di Pavia, via Taramelli 3b, I-27100 Pavia, ItalyFax: ϩ39 382 423108. E-mail: stoppini@unipv.it Abbreviations. Gdn/HCl, guanidine hydrochloride ; EM, electron microsc...

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