Characterization of Salt-Induced Oligomerization of Human β2-Microglobulin at Low pH

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• 作者：Dominic Narang ; Anubhuti Singh ; Hema M. Swasthi ; Samrat Mukhopadhyay
• 刊名：Journal of Physical Chemistry B
• 出版年：2016
• 出版时间：August 18, 2016
• 年：2016
• 卷：120
• 期：32
• 页码：7815-7823
• 全文大小：595K
• 年卷期：0
• ISSN：1520-5207

文摘

Misfolding and amyloid aggregation of human β₂-microglobulin (β₂m) have been linked to dialysis-related amyloidosis. Previous studies have shown that in the presence of different salt concentrations and at pH 2.5, β₂m assembles into aggregates with distinct morphologies. However, the structural and mechanistic details of the aggregation of β₂m, giving rise to different morphologies, are poorly understood. In this work, we have extensively characterized the salt-induced oligomers of the acid-unfolded state of β₂m using an array of biophysical tools including steady-state and time-resolved fluorescence, circular dichroism, dynamic light scattering, and atomic force microscopy imaging. Fluorescence studies using the oligomer-sensitive molecular rotor, 4-(dicyanovinyl)-julolidine, in conjunction with the light scattering and cross-linking assay indicated that at low salt (NaCl) concentrations β₂m exists as a disordered monomer, capable of transforming into ordered amyloid. In the presence of higher concentrations of salt, β₂m aggregates into a larger oligomeric species that does not appear to transform into amyloid fibrils. Site-specific fluorescence experiments using single Trp variants of β₂m revealed that the middle region of the protein is incorporated into these oligomers, whereas the C-terminal segment is highly exposed to bulk water. Additionally, stopped-flow kinetic experiments indicated that the formation of hydrophobic core and oligomerization occur concomitantly. Our results revealed the distinct pathways by which β₂m assembles into oligomers and fibrils.