Determination of the Absolute Molar Mass of [Fe-S]-Containing Proteins Using Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS)

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 at the 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.

Automated summary

Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) is a technique used to determine the absolute molar mass of macromolecules in solution by detecting their light scattering intensity. This technique can be applied to proteins and polymers of any shape without relying on assumptions or calibrations. However, corrections are needed for samples that absorb light at the MALS laser wavelength. In this study, various corrections were applied to determine the absolute molar mass of [2Fe-2S] and [4Fe-4S] cluster-containing proteins in both apo- and holo-forms. The determination of the absolute molar mass of the [2Fe-2S]-containing holo-NEET proteins revealed changes in oligomerization state upon cluster binding, highlighting an essential function of the cluster.