Determination of the Absolute Molar Mass of [Fe-S]-Containing Proteins Using Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS)
Language
EN
Article de revue
This item was published in
Biomolecules. 2022-02-09
English Abstract
Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) is a technique that determines the absolute molar mass (molecular weight) of macromolecules in solution, such as proteins or polymers, by ...Read more >
Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) is a technique that determines the absolute molar mass (molecular weight) of macromolecules in solution, such as proteins or polymers, by detecting their light scattering intensity. Because SEC-MALS does not rely on the assumption of the globular state of the analyte and the calibration of standards, the molar mass can be obtained for proteins of any shape, as well as for intrinsically disordered proteins and aggregates. Yet, corrections need to be made for samples that absorb light atthe wavelength of the MALS laser, such as iron–sulfur [Fe-S] cluster-containing proteins. We analyze several examples of [2Fe-2S] and [4Fe-4S] cluster-containing proteins, for which various corrections were applied to determine the absolute molar mass of both the apo- and holo-forms. Importantly, the determination of the absolute molar mass of the [2Fe-2S]-containing holo-NEET proteins allowed us to ascertain a change in the oligomerization state upon cluster binding and, thus, to highlight one essential function of the cluster.Read less <
English Keywords
SEC-MALS
size exclusion chromatography
multi-angle light scattering
molar mass
molecular weight
Fe-S cluster
iron-sulfur cluster
[Fe-S]-binding protein
NEET protein