Capillary electrophoresis investigation of a partially unfolded conformation of β2-microglobulin

Abstract

Dialysis-related amyloidosis is a disease in which partial unfolding of beta(2)-microglobulin plays a key pathogenetic role in the formation of the amyloid fibrils. We have recently demonstrated that a partially unfolded conformer of beta(2)-microglobulin is involved in fibrillogenesis and that this species is significantly populated under physiological conditions. In this work capillary electrophoresis has been used to measure the equilibrium between the native protein and this conformer in samples known to have a higher or lower amyloidogenic potential, namely full-length beta(2)-microglobulin, two truncated species and a mutant, created by replacing histidine in position 31 with thyrosine. In addition, for all protein species folding stability experiments have been carried out by monitoring the secondary structure by circular dichroism at increasing concentrations of guanidinium chloride. The values of free energy of unfolding in the absence of denaturant, obtained by elaboration of these experiments, were found to be inversely correlated to the area percent of the partially unfolded conformer, as measured by capillary electrophoresis. Affinity capillary electrophoresis experiments have been also carried out under nondenaturing conditions to assess the affinity of copper and suramin to either the native form or the conformational intermediate of full-length beta(2)-microglobulin.

Properties of Some Variants of Human β2-Microglobulin and Amyloidogenesis

“…Because the link between Cu 2ϩ binding and partial unfolding of ␤ 2 -m is the local charge increase on His-31, equivalent to acidification of a neutral ␤ 2 -m solution, to design a more stable protein we reasoned that a mutant His-313 Tyr might prevent the destabilizing imidazole titration, while preserving the local aromatic character. The expectation was successful as the designed mutant has been shown to display increased folding stability relative to the wild-type protein (Table I), along with reduced Cu 2ϩ binding (42). Although the His-31-mediated mechanism for Cu 2ϩ specificity was judged reasonable, an additional, alternative view was advanced involving a copper binding site in nonnative (unfolded) ␤ 2 -m states (32).…”

“…This latter nature of M I could be the case with ␤ 2 -m based on the NMR evidence (Figs. 2 and 9), despite the former possibility (thermodynamic intermediate) cannot be definitely ruled out, as also suggested by previous results (15,42). Once formed, OliN elongates by addition of M F , M I , or OliD and subsequent conformational rearrangement of the incorporated monomers.…”

“…The transient intermediate view we have just outlined is consistent with the previous reports concerning the occurrence of a monomeric ␤ 2 -m intermediate named I 2 and involved in fibrillogenesis (14,15,42). The I 2 intermediate was inferred from the analysis of the slow phase of the refolding kinetics and detected by circular dichroism and capillary electrophoresis at low protein concentration (1-40 M) always along the refolding pathway of ␤ 2 -m. The data obtained by real-time NMR (Fig.…”

“…2) suggest that I 2 corresponds to a poorly stable fold of the protein, with a dynamic hydrophobic packing pattern that seems, however, to conserve substantially the tertiary topology of native ␤ 2 -m and to convert slowly into the latter. The fibrillogenic propensity of I 2 that was observed on addition of seeding to refolding ␤ 2 -m solutions (15,42) is compatible with the NMR picture of a destabilized protein form undergoing an easier amyloidogenic transformation.…”

Recent advances in the study of biomolecular interactions by capillary electrophoresis

“…At increasing concentration of Cu 21 in the running buffer, a mobility shift was ovserved, accompanied by peak broadening for I 2 , whereas the N peak is unaffected. ACE permits to characterize the structural particularities of b 2 -m, providing useful insights for the development of new drugs [74]. ACE was used to detect a mobility shift in the presence of zinc ions binding to the highly basic nucleocapsid protein (NCp7) of HIV-1 [75].…”