Molecular Biophysics
Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry: αB-CRYSTALLIN DISSOCIATES β2-MICROGLOBULIN OLIGOMERS*

https://doi.org/10.1074/jbc.M112.448639Get rights and content
Under a Creative Commons license
open access

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β2-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aβ2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aβ2m. From analysis of the NMR spectra collected at various R3Aβ2m to αB-crystallin molar subunit ratios, it is concluded that the structured β-sheet core and the apical loops of R3Aβ2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type β2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type β2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated β2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing β2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation.

Background: β2-Microglobulin (β2m) is a paradigmatic amyloidogenic protein.

Results: In vitro, the molecular chaperone αB-crystallin affects the oligomerization and the fibrillogenesis of β2m and its R3A mutant.

Conclusion: αB-crystallin prevents β2m aggregation at various stages of its aggregation pathway.

Significance: Molecular chaperones may be relevant to amyloid formation in vivo.

Amyloid
Mass Spectrometry (MS)
Nuclear Magnetic Resonance
Protein Aggregation
Small Heat Shock Proteins
Amyloid Fibrils
NMR Spectroscopy
αB-crystallin
β2-Microglobulin
Native Mass Spectrometry

Cited by (0)

*

This work was supported by Italian Ministry of Education (MIUR) Grants PRIN N. 20083ERXWS, FIRB N. RBRN07BMCT, and FIRB N. RBFR109EOS, Fondazione Cariplo Project 2011-2096, Strategic Award MR/K000187/1, European Union Grant LSHM-CT-2006-037525-EURAMY, the National Health and Medical Research Council of Australia, and the Australian Research Council.

2

Both authors contributed equally to this work.

3

Present address: Institute for Molecular Biotechnology, Reinisch-Westfaelische Technische Hochschule Aachen University, 52074 Aachen, Germany.

4

Present address: Chemistry and Biochemistry Dept., University of California, La Jolla, CA 92037-0378.