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Abstract: Beta-2 microglobulin (β2m) is the light chain of class I major histocompatibility complex (MHC-I). β2m is an intrinsically amyloidogenic protein that can assemble into amyloid fibrils in a concentration dependent manner. β2m is accumulated in serum of haemodialysed patients, and deposited in the skeletal joints, causing dialysis related amyloidosis. Recent reports suggested that the loop comprised between β2m strands D and E is crucial for protein stability and for β2m propensity to aggregate as cross-β structured fibrils. In particular, the role of Trp60 for β2m stability has been highlighted by showing that the Trp60→Gly β2m mutant is more thermo-stable and less prone to aggregation than the wild type protein. On the contrary the Asp59→Pro β2m mutant shows lower Tm and stronger tendency to fibril aggregation. To further analyse such properties, the Trp60→Val β2m mutant has been expressed and purified; the propensity to fibrillar aggregation and the folding stability have been assessed, and the X-ray crystal structure determined to 1.8Å resolution. The W60V mutant structural features are discussed, focusing on the roles of the DE loop and of residue 60 in relation to β2m structure and its amyloid aggregation trends. [Copyright &y& Elsevier]

Abstract: β2-Microglobulin (β2m) is the light chain component of class I major histocompatibility complex (MHC-I). β2m is an intrinsically amyloidogenic protein that can assemble into amyloid fibrils in vitro and in vivo. Several recent reports suggested that the polypeptide loop comprised between β-strands D and E of β2m is important for protein stability and for the protein propensity to aggregate as amyloid fibrils. In particular, the roles of Trp60 for MHC-I assembly and β2m stability have been highlighted by showing that the β2m Trp60→Gly mutant is more stable and less prone to aggregation than the wild type protein. To further analyse such properties, the Trp60→Cys and Asp59→Pro β2m mutants have been expressed, purified, and their crystal structures determined. The stability to thermal denaturation and propensity to fibrillar aggregation have also been analysed. The experimental evidences gathered on the two mutants reinforce the hypothesis that conformational strain in the DE loop can affect β2m stability and amyloid aggregation properties. [Copyright &y& Elsevier]

Abstract: A variety of amyloid diseases are associated with fibrillar aggregates from N-terminal fragments of ApoA-I generated through a largely unexplored multi-step process. The understanding of the molecular mechanism is impaired by the lack of suitable amounts of the fibrillogenic polypeptides that could not be produced by recombinant methods so far. We report the production and the conformational analysis of recombinant ApoA-I 1–93 fragment. Similarly to the polypeptide isolated ex vivo, a pH switch from 7 to 4 induces a fast and reversible conformational transition to a helical state and leads to the identification of a key intermediate in the fibrillogenesis process. Limited proteolysis experiments suggested that the C-terminal region is involved in helix formation. The recombinant polypeptide generates fibrils at pH 4 on a time scale comparable with that of the native fragment. These findings open the way to studies on structural, thermodynamic, and kinetic aspects of ApoA-I fibrillogenesis. [Copyright &y& Elsevier]