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

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein beta(2)-microglobulin (beta 2m) and the molecular chaperone alpha B-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3A beta 2m was potently prevented by alpha B-crystallin. alpha B-crystallin also prevented the unfolding and nonfibrillar aggregation of R3A beta 2m. From analysis of the NMR spectra collected at various R3A beta 2m to alpha B-crystallin molar subunit ratios, it is concluded that the structured beta-sheet core and the apical loops of R3A beta 2m interact in a nonspecific manner with the alpha B-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type beta 2m at a 100-fold concentration excess with respect to alpha B-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type beta 2m oligomerization was effectively reduced by alpha B-crystallin. Furthermore, and most importantly, alpha B-crystallin reversibly dissociated beta 2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of alpha B-crystallin in preventing beta 2m 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.