Free-solution capillary electrophoresis for the structural characterization of proteins

Abstract

Proteins constitute a broad class of macromolecules which are abundant in all cells and are responsible for a wide variety of biological processes. Exploring the molecular mechanisms of these biological processes is pivotal, not only for understanding protein-related diseases and disorders, but also for the advancement of the biotechnological applications of proteins. Various techniques can be used for the structural characterization of proteins at both the atomic and molecular levels. Many proteins are structurally dynamic and thus may exhibit varying degrees of heterogeneity with regards to size, molar mass and molecular conformation. Currently, size-exclusion chromatography (SEC) coupled to multi-angle light scattering (MALS) is the most common technique used to study protein heterogeneity. Distributions of hydrodynamic volumes and molar masses can be obtained from SEC-MALS, however this technique faces several limitations such as high cost, poor reproducibility and adsorption, to name a few. Additionally, many proteins also undergo more subtle changes to their structures (such as domain rearrangements) which do not necessarily impact their overall size/molar mass. This project was therefore aimed at establishing a new method using free-solution capillary electrophoresis (CE) for the structural characterization of proteins, to be used complementarily to SEC. CE is a robust, cost- and time-effective technique that requires small sample amounts requiring minimal preparation. Additionally, the mechanism of separation enables distributions of electrophoretic mobilities to be obtained. CE in the critical conditions (CE-CC) allows for separation independent of molar mass, offering insight into the heterogeneity of a protein with regards to molecular conformations.

Date of Award	2018
Original language	English