The Complementarity of Nuclear Magnetic Resonance and Native Mass Spectrometry in Probing Protein-Protein Interactions.

Nuclear magnetic resonance (NMR) and native mass spectrometry (MS) are mature physicochemical techniques with long histories and important applications. NMR spectroscopy provides detailed information about the structure, dynamics, interactions, and chemical environment of biomolecules. MS is an effective approach for determining the mass of biomolecules with high accuracy, sensitivity, and speed. The two techniques offer unique advantages and provide solid tools for structural biology. In the present review, we discuss their individual merits in the context of their applications to structural studies in biology with specific focus on protein interactions and evaluate their limitations. We provide specific examples in which these techniques can complement each other, providing new information on the same scientific case. We discuss how the field may develop and what challenges are expected in the future. Overall, the combination of NMR and MS plays an increasingly important role in integrative structural biology, assisting scientists in deciphering the three-dimensional structure of composite macromolecular assemblies.

The plasma membrane-associated cation-binding protein PCaP1 of Arabidopsis thaliana is a uranyl-binding protein.

Uranium (U) is a naturally-occurring radionuclide that is toxic to living organisms. Given that proteins are primary targets of U(VI), their identification is an essential step towards understanding the mechanisms of radionuclide toxicity, and possibly detoxification. Here, we implemented a chromatographic strategy including immobilized metal affinity chromatography to trap protein targets of uranyl in Arabidopsis thaliana. This procedure allowed the identification of 38 uranyl-binding proteins (UraBPs) from root and shoot extracts. Among them, UraBP25, previously identified as plasma membrane-associated cation-binding protein 1 (PCaP1), was further characterized as a protein interacting in vitro with U(VI) and other metals using spectroscopic and structural approaches, and in planta through analyses of the fate of U(VI) in Arabidopsis lines with altered PCaP1 gene expression. Our results showed that recombinant PCaP1 binds U(VI) in vitro with affinity in the nM range, as well as Cu(II) and Fe(III) in high proportions, and that Ca(II) competes with U(VI) for binding. U(VI) induces PCaP1 oligomerization through binding at the monomer interface, at both the N-terminal structured domain and the C-terminal flexible region. Finally, U(VI) translocation in Arabidopsis shoots was affected in pcap1 null-mutant, suggesting a role for this protein in ion trafficking in planta.