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

Christophe Velours,Myriam Salameh,Nisha He,Béatrice Golinelli-Pimpaneau,Paolo Zecchin,Jingjing Zhou,Marie-Pierre Golinelli-Cohen

doi:10.3390/biom12020270

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 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.

Highlights

Is a technique that determines the absolute molar mass of macromolecules in solution, such as proteins or polymers, by detecting their light scattering intensity
We show that the molar mass of the holo-proteins can be determined more accurately than without the correction and that, in the case of two NEET proteins, the binding of the cluster triggers the dimerization of the proteins
We analyzed by size-exclusion chromatography (SEC)-MALS six [Fe-S]-binding proteins, two [2Fe-2S] regulatory proteins (CISD2 and mitoNEET) and four [4Fe-4S]-dependent enzymes

Summary

Introduction

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. Light Scattering (MALS) is an absolute technique that uses a collimated beam from a laser source to determine the exact mass in solution of proteins, lipids, detergents, nucleic acids, sugars or heterologous complexes and to evaluate their gyration radius [2–5]. Corrections of the light scattering measurements are required to determine the molar mass of polymers that are fluorescent or that absorb light at the operating laser wavelength. Whereas such corrections were previously described for lignin polymers [11], this topic is not frequently discussed in the literature.

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