Abstractβ-2-microglobulin (β2m) self-associates into fibrillar amyloid deposits in the musculoskeletal system of patients undergoing hemodialysis treatment. Previous studies have shown that stoichiometric amounts of Cu(II) at near physiological conditions can cause β2m to organize into native-like dimers prior to forming amyloid fibrils. Here, we report the results from selective covalent labeling reactions combined with mass spectrometry that provide insight into the amino acid residues that mediate dimer formation in the wild-type protein. Using three complementary covalent labeling reagents, we find that the dimer interface is formed by the anti-parallel stacking of ABED β-sheets from two β2m monomers. In addition, our data clearly indicate that a dimer interface involving the interactions of D-D strands from separate protein units as seen in the recent crystal structures of two mutant β2m oligomers is unlikely.β-2-microglobulin (β2m) is the non-covalently bound light chain of the class I major histocompatibility complex (MHC-I) (1). It is a monomeric protein with 99 residues (~12 kDa). It adopts a seven-stranded β-sandwich fold with one β sheet formed by four strands and the other by three strands. A disulfide bond between Cys25 and Cys80 links strands the two sheets in the folded state of the protein. β2m is vital for the correct folding, assembly, and cell-surface expression of the MHC-I complex. As part of normal cell turnover, β2m is released from the MHC-I complex and carried to the kidney where it is degraded. Upon renal failure, serum levels of β2m increase up to ~60 times above the normal level of 0.1 µM, and the protein aggregates into insoluble amyloid fibrils in the joints (2,3). Elevated β2m concentrations alone, however, are not sufficient to trigger fibrillogenesis (4,5). β2m amyloid formation must therefore result from features unique to hemodialysis, but the exact cause in vivo is not known. ®2m amyloid fibrils can be generated in vitro, though, under acidic conditions (pH < 3.6) (6), by removing the first six N-terminal amino acids (7), by mixing the protein with collagen at pH = 6.4 (8), by sonicating the protein in the presence of sodium dodecyl sulfate at pH = *Corresponding author Department of Chemistry, University of Massachusetts, Amherst, rwvachet@chem.umass.edu, Telephone: (413) 545-2733, Fax: (413) . † Department of Chemistry, Universidad Industrial de Santander, AA 678, Bucaramanga, Colombia
SUPPORTING INFORMATION AVAILABLEAdditional data can be found in the Supporting Information. These data include (i) plots showing the intensities of unmodified and modified forms of fragment Ile1-Tyr10 for three trials in the absence of Cu; (ii) tables presenting the intensities of unmodified and modified forms of peptide fragments containing residues Asn83, Lys94, and Lys6; (iii) plots illustrating the intensities of unmodified and modified forms of fragment Ile1-Tyr10 in the absence of Cu and 2 hours after addition of Cu; (iv) plots illustrating the extent of NHSA modification of β2m res...
