• Amyloid-forming peptides from beta2-microglobulin-Insights into the mechanism of fibril formation in vitro.

      Jones, Susan; Kad, N.M.; Manning, J.; Radford, S.E. (2003)
      ß2-Microglobulin (ß2m) is one of over 20 proteins known to be involved in human amyloid disease. Peptides equivalent to each of the seven ß-strands of the native protein, together with an eighth peptide (corresponding to the most stable region in the amyloid precursor conformation formed at pH 3.6, that includes residues in the native strand E plus the eight succeeding residues (named peptide E¿)), were synthesised and their ability to form fibrils investigated. Surprisingly, only two sequences, both of which encompass the region that forms strand E in native ß2m, are capable of forming amyloid-like fibrils in vitro. These peptides correspond to residues 59¿71 (peptide E) and 59¿79 (peptide E¿) of intact ß2m. The peptides form fibrils under the acidic conditions shown previously to promote amyloid formation from the intact protein (pH <5 at low and high ionic strength), and also associate to form fibrils at neutral pH. Fibrils formed from these two peptides enhance fibrillogenesis of the intact protein. No correlation was found between secondary structure propensity, peptide length, pI or hydrophobicity and the ability of the peptides to associate into amyloid-like fibrils. However, the presence of a relatively high content of aromatic side-chains correlates with the ability of the peptides to form amyloid fibrils. On the basis of these results we propose that residues 59¿71 may be important in the self-association of partially folded ß2m into amyloid fibrils and discuss the relevance of these results for the assembly mechanism of the intact protein in vitro.
    • Role of the N- and C-terminal strands of beta 2-microglobulin in amyloid formation at neutral pH.

      Jones, Susan; Smith, D.P.; Radford, S.E. (2003)
      Beta 2-microglobulin (ß2m) is known to form amyloid fibrils de novo in vitro under acidic conditions (below pH 4.8). Fibril formation at neutral pH, however, has only been observed by deletion of the N-terminal six residues; by the addition of pre-assembled seeds; or in the presence of Cu2+. Based on these observations, and other structural data, models for fibril formation of ß2m have been proposed that involve the fraying of the N and C-terminal ß-strands and the consequent loss of edge strand protective features. Here, we examine the role of the N and C-terminal strands in the initiation of fibrillogenesis of ß2m by creating point mutations in strands A and G and comparing the properties of the resulting proteins with variants containing similar mutations elsewhere in the protein. We show that truncation of buried hydrophobic side-chains in strands A and G promotes rapid fibril formation at neutral pH, even in unseeded reactions, and increases the rate of fibril formation under acidic conditions. By contrast, similar mutations created in the remaining seven ß-strands of the native protein have little effect on the rate or pH dependence of fibril formation. The data are consistent with the view that perturbation of the N and C-terminal edge strands is an important feature in the generation of assembly-competent states of ß2m.
    • A Systematic Investigation into the Effect of Protein Destabilisation on Beta 2-Microglobulin Amyloid Formation

      Jones, Susan; Smith, D.P.; Serpell, L.C; Sunde, M.; Radford, S.E. (2009-07-20)
      Beta-2-microglobulin (2m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (~1 m), straight morphology are produced by incubation of the protein at pH=<3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of 2m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states.
    • A Systematic study of the effect of physiological factors on beta2-microglobulin amyloid formation at neutral pH

      Jones, Susan; Myers, S.L.; Radford, S.E.; Tennent, G.A. (2006)
      ß2-microglobulin (ß2m) forms amyloid fibrils that deposit in the musculo-skeletal system in patients undergoing long-term hemodialysis. How ß2m self-assembles in vivo is not understood, since the monomeric wild-type protein is incapable of forming fibrils in isolation in vitro at neutral pH, while elongation of fibril-seeds made from recombinant protein has only been achieved at low pH or at neutral pH in the presence of detergents or cosolvents. Here we describe a systematic study of the effect of 11 physiologically relevant factors on ß2m fibrillogenesis at pH 7.0 without denaturants. By comparing the results obtained for the wild-type protein with those of two variants (¿N6 and V37A), the role of protein stability in fibrillogenesis is explored. We show that ¿N6 forms low yields of amyloid-like fibrils at pH 7.0 in the absence of seeds, suggesting that this species could initiate fibrillogenesis in vivo. By contrast, high yields of amyloid-like fibrils are observed for all proteins when assembly is seeded with fibril-seeds formed from recombinant protein at pH 2.5 stabilized by the addition of heparin, serum amyloid P component (SAP), apolipoprotein E (apoE), uremic serum, or synovial fluid. The results suggest that the conditions within the synovium facilitate fibrillogenesis of ß2m and show that different physiological factors may act synergistically to promote fibril formation. By comparing the behavior of wild-type ß2m with that of ¿N6 and V37A, we show that the physiologically relevant factors enhance fibrillogenesis by stabilizing fibril-seeds, thereby allowing fibril extension by rare assembly competent species formed by local unfolding of native monomers.