Using Capillary Electrophoresis to Investigate Protein Conformational and Compositional Heterogeneity.

Autor: Grosas AB; Research School of Chemistry, The Australian National University, Acton, ACT 2601, Australia.; Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW 2552, Australia.; Australian Research Council Centre for Cryo-electron Microscopy of Membrane Proteins, University of Wollongong, Wollongong, NSW 2522, Australia., Du Plessis MD; Australian Centre for Research on Separation Science (ACROSS), Western Sydney University, School of Science, Parramatta, NSW 2150, Australia., Thevarajah JJ; Australian Centre for Research on Separation Science (ACROSS), Western Sydney University, School of Science, Parramatta, NSW 2150, Australia., Gaborieau M; Australian Centre for Research on Separation Science (ACROSS), Western Sydney University, School of Science, Parramatta, NSW 2150, Australia., Carver JA; Research School of Chemistry, The Australian National University, Acton, ACT 2601, Australia., Castignolles P; Australian Centre for Research on Separation Science (ACROSS), Western Sydney University, School of Science, Parramatta, NSW 2150, Australia.; Sorbonne Universite, Institut Parisien de Chimie Moleculaire, Equipe Chimie des Polymeres, Paris, France.
Jazyk: angličtina
Zdroj: Chembiochem : a European journal of chemical biology [Chembiochem] 2024 Jun 03; Vol. 25 (11), pp. e202400108. Date of Electronic Publication: 2024 May 16.
DOI: 10.1002/cbic.202400108
Abstrakt: Detailed insights into protein structure/function relationships require robust characterization methodologies. Free-solution capillary electrophoresis (CE) is a unique separation technique which is sensitive to the conformation and/or composition of proteins, and therefore provides information on the heterogeneity of these properties. Three unrelated, conformationally/compositionally-altered proteins were separated by CE. An electrophoretic mobility distribution was determined for each protein along with its conformational and/or compositional heterogeneity. The CE results were compared with molar mass distributions obtained from size-exclusion chromatography coupled to light scattering (SEC-MALS). Bovine serum albumin multimers and two monomeric species were separated, highlighting variations in conformational/compositional heterogeneity among the multimers. Analysis of yeast alcohol dehydrogenase resolved two monomeric conformers and various tetrameric species, illustrating the impact of zinc ion removal and disulfide bond reduction on the protein's heterogeneity. The apo (calcium-free) and holo forms of bovine α-lactalbumin were separated and differences in the species' heterogeneity were measured; by contrast, the SEC-MALS profiles were identical. Comparative analysis of these structurally unrelated proteins provided novel insights into the interplay between molar mass and conformational/compositional heterogeneity. Overall, this study expands the utility of CE by demonstrating its capacity to discern protein species and their heterogeneity, properties which are not readily accessible by other analytical techniques.
(© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)
Databáze: MEDLINE
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