NMR-based characterization of a refolding intermediate of β2-microglobulin labeled using a wheat germ cell-free system

In patients with dialysis-related amyloidosis, beta 2-microglobulin (beta 2-m) is a major structural component of amyloid fibrils. It has been suggested that the partial unfolding of beta 2-m is a prerequisite to the formation of amyloid fibrils, and that the folding intermediate trapped by the non-native trans-Pro32 isomer leads to the formation of amyloid fibrils. Although clarifying the structure of this refolding intermediate by high resolution NMR spectroscopy is important, this has been made difficult by the limited lifetime of the intermediate. Here, we studied the structure of the refolding intermediate using a combination of amino acid selective labeling with wheat germ cell-free protein synthesis and NMR techniques. The HSQC spectra of beta 2-ms labeled selectively at either phenylalanine, leucine, or valine enabled us to monitor the structures of the refolding intermediate. The results suggested that the refolding intermediate has an overall fold and cores similar to the native structure, but contains disordered structures around Pro32. The fluctuation of the beta-sheet regions especially the last half of the beta B strand and the first half of the beta E strand, both suggested to be important for amyloidogenicity, may transform beta 2-m into an amyloidogenic structure.