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NMR‐based characterization of a refolding intermediate of β2‐microglobulin labeled using a wheat germ cell‐free system
Author(s) -
Kameda Atsushi,
Morita EugeneHayato,
Sakurai Kazumasa,
Naiki Hironobu,
Goto Yuji
Publication year - 2009
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.179
Subject(s) - chemistry , fibril , amyloid (mycology) , beta 2 microglobulin , heteronuclear single quantum coherence spectroscopy , nuclear magnetic resonance spectroscopy , folding (dsp implementation) , valine , protein folding , amyloidosis , amino acid , amyloid fibril , crystallography , biochemistry , stereochemistry , biology , amyloid β , medicine , inorganic chemistry , disease , pathology , electrical engineering , immunology , engineering
In patients with dialysis‐related amyloidosis, β2‐microglobulin (β2‐m) is a major structural component of amyloid fibrils. It has been suggested that the partial unfolding of β2‐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 β2‐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 β‐sheet regions especially the last half of the βB strand and the first half of the βE strand, both suggested to be important for amyloidogenicity, may transform β2‐m into an amyloidogenic structure.