Molecular Insights into the Role of Serum Amyloid-P Component in the Stabilzation of Fibrillar Beta-2 Microglobulin

Serum amyloid-P component is found ubiquitously in amyloid deposits and has been shown to stabilize fibrillar structures and prevent clearance by the host's defences. Here we report on solid-state NMR studies on fibrillar deposits formed from β2-microglobulin, typically found in patients with dialysis related amyloidosis, and their interactions with serum amyloid-P component.We have successfully undertaken the expression, purification and refolding of the 99 residue β2-microglobulin and established conditions for optimal binding of serum amyloid-P component. High resolution solid-state magic-angle spinning (MAS)-NMR spectra obtained from the fibrils indicate that within the fibrils the β2-microglobulin adopts a homogeneous structure. Using two-dimension homo- and hetero-nuclear correlation spectroscopy we have been able to assign several of the sites within the protein. Currently we are using a range of labelling schemes and acquiring three-dimensional data-sets to complete this assignment. Comparison of the assignment with that obtained from monomeric β2-microglobulin in solution is beginning to provide valuable insights into the structural changes occurring upon fibrilization. Similar comparisons with fibres decorated with serum amyloid-P component should provide valuable insights into how this molecule interacts and stabilizes amyloid fibrils at a molecular level.During the course of these experiments we have also obtained 2D correlation data on inclusion bodies of β2-microglobulin. The resolution attained is not as high as that observed in the fibrillar spectra, however they permit the assignment of resonances to amino acids with the β2-microglobulin. This suggests that within the inclusion bodies the β2-microglobulin adopts a well defined conformation with the lower spectral resolution arising from reduction in dynamics in these highly packed structures. Detailed comparisons of the data with that obtained from the soluble and fibrillar β2-microglobulin should provide insights into the nature of this structure.