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Capillary electrophoresis investigation of a partially unfolded conformation of beta(2)-microglobulin
Ersilia De Lorenzi1, Silvia Grossi, Gabriella Massolini
1Department of Pharmaceutical Chemistry, University of Pavia, Viale Taramelli 12, I-27100 Pavia, Italy. ersidelo@unipv.it
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.
Insights
Dialysis-related amyloidosis involves beta(2)-microglobulin unfolding. This study quantifies the partially unfolded protein, linking its levels to disease risk and stability.
Area of Science:
- Biochemistry
- Protein Misfolding Diseases
- Amyloidosis
Background:
- Dialysis-related amyloidosis (DRA) is linked to beta(2)-microglobulin (β2M) partial unfolding.
- A partially unfolded β2M conformer is implicated in amyloid fibril formation.
- This intermediate species is present under physiological conditions.
Purpose of the Study:
- To quantify the equilibrium between native and partially unfolded β2M.
- To correlate protein stability and conformational intermediate population with amyloidogenic potential.
- To investigate the binding of copper and suramin to different β2M conformers.
Main Methods:
- Capillary electrophoresis (CE) to measure native vs. unfolded β2M equilibrium.
- Circular dichroism (CD) spectroscopy to assess protein folding stability via guanidinium chloride denaturation.
- Affinity CE under non-denaturing conditions to determine ligand binding.
Main Results:
- CE successfully quantified the partially unfolded β2M conformer across different protein variants.
- Folding stability (free energy of unfolding) inversely correlated with the population of the partially unfolded conformer.
- Affinity CE revealed differential binding of copper and suramin to native and intermediate β2M forms.
Conclusions:
- The population of the partially unfolded β2M conformer is a key determinant of amyloidogenic potential in DRA.
- Protein stability directly influences the propensity for forming disease-associated amyloid fibrils.
- Understanding β2M conformational dynamics and ligand interactions is crucial for DRA research.
