The role of conformational flexibility in β₂‐microglobulin amyloid fibril formation at neutral pH

Abstract

RATIONALE Amyloid formation is implicated in a number of human diseases. β2-microglobulin (β2m) is the precursor protein in dialysis-related amyloidosis and it has been shown that partial, or more complete, unfolding is key to amyloid fibril formation in this pathology. Here the relationship between conformational flexibility and β2m amyloid formation at physiological pH has been investigated.METHODS HDX-ESI-MS was used to study the conformational dynamics of β2m. Protein engineering, or the addition of Cu2+ ions, sodium dodecyl sulphate, trifluoroethanol, heparin, or protein stabilisers, was employed to perturb the conformational dynamics of β2m. The fibril-forming propensities of the protein variants and the wild-type protein in the presence of additives, which resulted in >5-fold increase in the EX1 rate of HDX, were investigated further.RESULTS ESI-MS revealed that HDX occurs via a mixed EX1/EX2 mechanism under all conditions. Urea denaturation and tryptophan fluorescence indicated that EX1 exchange occurred from a globally unfolded state in wild-type β2m. Although >30-fold increase in the HDX exchange rate was observed both for the protein variants and for the wild-type protein in the presence of specific additives, large increases in exchange rate did not necessarily result in extensive de novo fibril formation.CONCLUSIONS The conformational dynamics measured by the EX1 rate of HDX do not predict the ability of β2m to form amyloid fibrils de novo at neutral pH. This suggests that the formation of amyloid fibrils from β2m at neutral pH is dependent on the generation of one or more specific aggregation-competent species which facilitate self-assembly. Copyright © 2012 John Wiley & Sons, Ltd.

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

“…By massively increasing its interest in protein therapeutics within these last years, the biopharmaceutical industry became particularly interested in improving and developing spatially resolved analytical techniques like HDX-MS for its needs in both research and quality control. (14,15) Since so far HDX-MS was mostly implemented on monomeric proteins (16)(17)(18)(19)(20)(21)(22) and only a few studies have been conducted on homo-oligomeric proteins (23)(24)(25)(26)(27) or protein complexes, (28)(29)(30)(31) efforts have to be continued for extracting the dynamic determinants of complexes.…”

Expanding the Repertoire of Amyloid Polymorphs by Co-polymerization of Related Protein Precursors

“…Despite truncation of the N-terminal six residues, ΔN6 displays only minor structural differences compared with hβ 2 m in the native form (25). Although the structural properties of ΔN6 cannot explain its enhanced ability to form amyloid fibrils at neutral pH, increased conformational dynamics evidenced by NMR relaxation times ( T 2 values) (25), hydrogen exchange protection (27–29), molecular dynamics simulations (30), and denaturation with guanidinium chloride (GuHCl) (31) have been linked to its ability to form fibrils at this pH.…”

Assessing the Effect of Loop Mutations in the Folding Space of β2-Microglobulin with Molecular Dynamics Simulations

“…Indeed, experiments in vitro have shown that amyloid fibrils will not form in physiological conditions (of pH and temperature) even when the concentration of Hβ 2 m largely exceeds that observed in DRA patients [5,6]. Although it is widely recognized that WT Hβ 2 m is unable to efficiently nucleate fibrillogenesis in vitro under physiological conditions, it is also known that a multitude of environmental factors (e.g., acidic pH [7], agitation [8], addition of Cu 2+ in the presence of urea [9], co-solvents [10]), can trigger the formation of β 2 m amyloid fibrils in laboratory experiments.…”