title: NMR-based characterization of a refolding intermediate of beta2-microglobulin labeled using a wheat germ cell-free system.
abstract: In patients with dialysis-related amyloidosis, beta2-microglobulin (beta2-m) is a major structural component of amyloid fibrils. It has been suggested that the partial unfolding of beta2-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 beta2-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 betaB strand and the first half of the betaE strand, both suggested to be important for amyloidogenicity, may transform beta2-m into an amyloidogenic structure.
authors.affiliation: Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kameda
authors.forename: Eugene-Hayato
authors.initials: EH
authors.lastname: Morita
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Sakurai
authors.forename: Hironobu
authors.initials: H
authors.lastname: Naiki
authors.forename: Yuji
authors.initials: Y
authors.lastname: Goto
mesh_terms: D000682:Amyloid / Q000737:chemistry; D002474:Cell-Free System; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D014908:Triticum / Q000737:chemistry; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D000682:Amyloid; D001613:beta 2-Microglobulin