Interactions of charged ligands with β2-microglobulin conformers in affinity capillary electrophoresis

Alternative conformations of β2-microglobulin (β2m) are involved in its transformation from soluble monomeric precursor molecules to the insoluble polymeric material that constitutes β2m amyloid. Accordingly, non-native conditions such as low pH or high ionic strength promote β2m amyloid formation in vitro. The early events in these processes are not well known, partly because of the paucity of techniques available for the characterization of transient folding intermediates in proteins. We have used high-resolution separations in capillaries (capillary electrophoresis, CE) to resolve putative conformer fractions in native and structurally modified β2m and to show the induction of alternatively folded β2m under different experimental conditions. The conformer fractions are observed as distinct peaks in the separation profiles and thus it is possible to probe for the reactivity of these individual β2m species with specific ligands that, upon binding, alter analyte mobility in affinity capillary electrophoresis experiments. Interactions were shown in this way for the negatively charged substances heparin, Congo red, and suramin, as well as for Cu2+ ions. Marked differences in the binding behavior of the β2m conformational variants compared with native β2m could be demonstrated. This approach for conformer separation and binding characterization is a valuable starting point for the assessment of various ligand molecules, or analogues thereof, as agents capable of perturbing the mechanisms of fibril formation.