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Abstract
The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.
Highlights
SPL2 is a 383-amino-acid-long protein localized to the outer membrane of chloroplasts
A part of the linker shares partial sequence similarity with lanthanide-binding peptides, as suggested by multiple sequence alignments (Figure 1A). This analysis indicated that SPL2 affinity for Ln ions might not be as high as in the case of the iteratively designed and tested lanthanide-binding tags (LBT) shown in
Encouraged by preliminary luminescence data that provided the first evidence for interactions between Tb3+ and SPL2cyt, we investigated metal binding by complementary techniques such as circular dichroism (CD)
Summary
Lanthanide probes and lanthanide-binding tags (LBT) present attractive photophysical and magnetic properties for cellular biologists and biophysicists. Their significant advantages over conventional fluorophores are not limited to their unique emission profiles or extremely long-lived luminescence [1] but extend to their resistance to photobleaching and the specificity of the labeling. The small size of LBTs is of paramount importance as it results in a low probability of adverse effects exerted on protein functions
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