Nucleotide hydrolases play integral yet poorly understood roles in several metallocluster biosynthetic pathways. For example, the cytosolic iron-sulfur cluster assembly (CIA) is initiated by the CIA scaffold, an ATPase which builds new iron-sulfur clusters for proteins localized to the cytosol and the nucleus in eukaryotic organisms. While in vivo studies have demonstrated the scaffold's nucleotide hydrolase domain is vital for its function, in vitro approaches have not revealed tight allosteric coupling between the cluster scaffolding site and the ATPase site. Thus, the role of ATP hydrolysis has been hard to pinpoint. Herein, we describe methods to probe the nucleotide affinity and hydrolysis activity of the CIA scaffold from yeast, which is comprised of two homologous polypeptides called NBP35 and CFD1. In particular, we report two different equilibrium binding assays that make use of commercially available fluorescent nucleotide analogs. Importantly, these assays can be applied to probe nucleotide affinity of both the apo- and holo-forms of the CIA scaffold. Generally, these fluorescent nucleotide analogs have been underutilized to probe metal trafficking NTPase because one of the most commonly used probes, mantATP, which is labeled with the methylanthraniloyl probe via the 2' or 3' sugar hydroxyls, has an absorption which overlaps with the UV-Vis features of many metal-binding proteins. However, by exploiting analogs like BODIPY-FL and trinitrophenyl-labeled nucleotides which have better photophysical properties for metalloprotein applications, these approaches have the potential to reveal the mechanistic underpinnings of NTPases required for metallocluster biosynthesis.

• PMID: 29746244
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Journal Article | Research Support, U.S. Gov't, Non-P.H.S.

Authors

Grossman JD, Camire EJ, Perlstein DL

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