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...

The main pathogenic process underlying dialysis-related amyloidosis (DRA) is the accumulation of β-2-microglobulin (β2m) as amyloid fibrils in the musculoskeletal system, and some evidence suggests that Cu(II) may play a role in β2m amyloid formation. Cu(II)-induced β2m fibril formation is preceded by the formation of discrete, oligomeric intermediates, including dimers, tetramers, and hexamers. In this work, we use selective covalent labeling reactions combined with mass spectrometry to investigate the amino acids responsible for mediating tetramer formation in wild-type β2m. By comparing the labeling patterns of the monomer, dimer, and tetramer, we find evidence that the tetramer interface is formed by the interaction of D strands from one dimer unit and G strands from another dimer unit. This covalent labeling data along with molecular dynamics calculations enable the construction of a tetramer model that indicates how the protein might proceed to form even higher order oligomers.

Nonequilibrium molecular dynamics simulations show that steady-state systems obtained by microwave heating are qualitatively different from those at thermal equilibrium. This difference arises because energy transfer from hotter to colder species is not efficient enough to equilibrate the distribution of energy. Under nonequilibrium conditions, we found that microwave radiation can selectively heat methanol in a binary mixture of methanol-benzene adsorbed in faujasite zeolite. The difference in steady-state temperatures follows the trend Tmethanol > Tbenzene > Tzeolite, which is qualitatively consistent with recent experimental results.

We have applied electronic structure methods to the calculation of transition state parameters for the O͑1͒→O͑4͒ proton transfer in H-Y zeolite. We arrive at a set of recommendations for calculating these transition state parameters accurately and efficiently. Density functional theory using the B3LYP functional and basis sets of triple-quality in the valence space, and including polarization functions on all atoms, is the most efficient method for converging structures and vibrational frequencies. For converging classical barrier heights, we find it necessary to augment MP2 barrier heights calculated using large basis sets with MP4 energies obtained in more limited basis sets. We obtain an O͑1͒→O͑4͒ barrier height of 86.1 kJ mol Ϫ1 , and find the curvature of the barrier at the transition state to be 1570 cm Ϫ1. Including long range effects from the work of Sauer et al. ͓ACS Symp. Ser. 721, 358 ͑1999͔͒ results in a higher barrier, which we estimate to be 97.1 kJ mol Ϫ1. We attribute the fact that our barriers are significantly larger than those reported in the experimental literature to the neglect of tunneling in the interpretation of experimental data.

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