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Updated: Jun 21, 2026
Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)
Published on: November 2, 2018
NMR-based characterization of a refolding intermediate of beta2-microglobulin labeled using a wheat germ cell-free
Atsushi Kameda1, Eugene-Hayato Morita, Kazumasa Sakurai
1Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
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.
Insights
Dialysis-related amyloidosis involves beta2-microglobulin (beta2-m) forming amyloid fibrils. This study reveals the refolding intermediate has a native-like fold but disordered regions, potentially driving amyloid formation.
Area of Science:
- Biochemistry
- Structural Biology
- Medical Research
Background:
- Dialysis-related amyloidosis is linked to beta2-microglobulin (beta2-m) amyloid fibril formation.
- Partial unfolding of beta2-m and a non-native trans-Pro32 isomer are implicated in amyloidogenesis.
Purpose of the Study:
- To elucidate the structure of the beta2-m refolding intermediate.
- To understand the structural basis of beta2-m amyloidogenicity.
Main Methods:
- Utilized wheat germ cell-free protein synthesis for amino acid selective labeling.
- Employed Nuclear Magnetic Resonance (NMR) spectroscopy, specifically HSQC spectra, to monitor intermediate structures.
- Investigated selectively labeled beta2-m at phenylalanine, leucine, and valine residues.
Main Results:
- The refolding intermediate exhibits an overall fold and core structure similar to native beta2-m.
- Disordered structures were identified around the Pro32 residue in the intermediate.
- Fluctuations in beta-sheet regions (betaB and betaE strands) were observed, potentially promoting amyloidogenic transformation.
Conclusions:
- The refolding intermediate of beta2-m possesses a largely native-like structure with localized disorder around Pro32.
- Dynamic changes in specific beta-sheet regions may be critical for the transition of beta2-m into an amyloidogenic state.
- This structural insight aids in understanding the pathogenesis of dialysis-related amyloidosis.
