title: Monitoring the interaction between β2-microglobulin and the molecular chaperone αB-crystallin by NMR and mass spectrometry: αB-crystallin dissociates β2-microglobulin oligomers.
abstract: 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.
authors: Dipartimento di Scienze Mediche e Biologiche, Università di Udine, 33100 Udine, Italy.
authors: Gennaro
authors: G
authors: Esposito
authors: Megan
authors: M
authors: Garvey
authors: Vera
authors: V
authors: Alverdi
authors: Fabio
authors: F
authors: Pettirossi
authors: Alessandra
authors: A
authors: Corazza
authors: Federico
authors: F
authors: Fogolari
authors: Maurizio
authors: M
authors: Polano
authors: P Patrizia
authors: PP
authors: Mangione
authors: Sofia
authors: S
authors: Giorgetti
authors: Monica
authors: M
authors: Stoppini
authors: Agata
authors: A
authors: Rekas
authors: Vittorio
authors: V
authors: Bellotti
authors: Albert J R
authors: AJ
authors: Heck
authors: John A
authors: JA
authors: Carver
mesh_terms: D000682:Amyloid / Q000737:chemistry; D052160:Benzothiazoles; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D055550:Protein Stability; D021241:Spectrometry, Mass, Electrospray Ionization; D013844:Thiazoles / Q000737:chemistry; D038203:alpha-Crystallin B Chain / Q000737:chemistry*; D001613:beta 2-Microglobulin / Q000737:chemistry*
chemical_list: D000682:Amyloid; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D013844:Thiazoles; D038203:alpha-Crystallin B Chain; D001613:beta 2-Microglobulin; C009462:thioflavin T
keywords: Amyloid; Amyloid Fibrils; Mass Spectrometry (MS); NMR Spectroscopy; Native Mass Spectrometry; Nuclear Magnetic Resonance; Protein Aggregation; Small Heat Shock Proteins; αB-crystallin; β2-Microglobulin
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