Determination of the Absolute Molar Mass of [Fe-S]-Containing Proteins Using Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS)
Christophe Velours,
Jingjing Zhou,
Paolo Zecchin,
Nisha He,
Myriam Salameh,
Marie-Pierre Golinelli-Cohen,
Béatrice Golinelli-Pimpaneau
Affiliations
Christophe Velours
Fundamental Microbiology and Pathogenicity Laboratory, UMR 5234 CNRS-University of Bordeaux, SFR TransBioMed, 33076 Bordeaux, France
Jingjing Zhou
Laboratoire de Chimie des Processus Biologiques, UMR 8229 CNRS, Collège de France, Sorbonne Université, 11 Place Marcelin Berthelot, 75231 Paris, France
Paolo Zecchin
Laboratoire de Chimie des Processus Biologiques, UMR 8229 CNRS, Collège de France, Sorbonne Université, 11 Place Marcelin Berthelot, 75231 Paris, France
Nisha He
Laboratoire de Chimie des Processus Biologiques, UMR 8229 CNRS, Collège de France, Sorbonne Université, 11 Place Marcelin Berthelot, 75231 Paris, France
Myriam Salameh
Institut de Chimie des Substances Naturelles, Université Paris-Saclay, CNRS, UPR2301, 91198 Gif-sur-Yvette, France
Marie-Pierre Golinelli-Cohen
Institut de Chimie des Substances Naturelles, Université Paris-Saclay, CNRS, UPR2301, 91198 Gif-sur-Yvette, France
Béatrice Golinelli-Pimpaneau
Laboratoire de Chimie des Processus Biologiques, UMR 8229 CNRS, Collège de France, Sorbonne Université, 11 Place Marcelin Berthelot, 75231 Paris, France
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.
