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Keywords:

  • Amyloid fibrils;
  • copper binding;
  • dialysis-related amyloidosis;
  • heteronuclear NMR;
  • hydrogen/deuterium exchange;
  • β2-microglobulin;
  • protein folding/misfolding
  • β2-m, β2-microglobulin;
  • CD, circular dichroism;
  • H/D, hydrogen/deuterium;
  • HSQC, heteronuclear single quantum coherence;
  • NMR, nuclear magnetic resonance;
  • NOE, η, nuclear Overhauser effect;
  • R1, longitudinal relaxation rates;
  • TOCSY, total correlation spectroscopy

Abstract

A key pathological event in dialysis-related amyloidosis is the fibril formation of β2-microglobulin (β2-m). Because β2-m does not form fibrils in vitro, except under acidic conditions, predisposing factors that may drive fibril formation at physiological pH have been the focus of much attention. One factor that may be implicated is Cu2+ binding, which destabilizes the native state of β2-m and thus stabilizes the amyloid precursor. To address the Cu2+-induced destabilization of β2-m at the atomic level, we studied changes in the conformational dynamics of β2-m upon Cu2+ binding. Titration of β2-m with Cu2+ monitored by heteronuclear NMR showed that three out of four histidines (His13, His31, and His51) are involved in the binding at pH 7.0. 1H-15N heteronuclear NOE suggested increased backbone dynamics for the residues Val49 to Ser55, implying that the Cu2+ binding at His51 increased the local dynamics of β-strand D. Hydrogen/deuterium exchange of amide protons showed increased flexibility of the core residues upon Cu2+ binding. Taken together, it is likely that Cu2+ binding increases the pico- to nanosecond fluctuation of the β-strand D on which His51 exists, which is propagated to the core of the molecule, thus promoting the global and slow fluctuations. This may contribute to the overall destabilization of the molecule, increasing the equilibrium population of the amyloidogenic intermediate.