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bioRxiv · 10.1101/820670

A microplate screen for metal-binding activity based on a nickel-binding protein from Clostridium carboxidivorans

Diep, P.·Mahadevan, R.·Yakunin, A.
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Abstract

Solute-binding proteins (SBPs) from ATP-binding cassette (ABC) transporters play crucial roles across all forms of life in transporting compounds against chemical gradients. Some SBPs have evolved to scavenge metal substrates from the environment with nanomolar and micromolar affinities (KD). There exist well established techniques like isothermal titration calorimetry for thoroughly studying these metalloprotein interactions with metal ions, but they are low-throughput. For protein libraries comprised of many metalloprotein homologues and mutants, and for collections of buffer conditions and potential ligands, the throughput of these techniques is paramount. In this study, we describe an improved method termed the microITFQ-LTA and validated it using CjNikZ, a well-characterized nickel-specific SBP (Ni-BP) from Campylobacter jejuni. We then demonstrated how the microITFQ-LTA can be designed to screen through a small collection of buffers and ligands to elucidate the binding profile of a putative Ni-BP from Clostridium carboxidivorans that we call CcSBPII. Through this study, we showed CcSBPII can bind to various metal ions with KD ranged over 3 orders of magnitude. In the presence of L-histidine, CcSBPII could bind to Ni2+ over 2000-fold more tightly, which was 11.6-fold tighter than CjNikZ given the same ligand. HighlightsO_LIan improved version of a high-throughput screen (microITFQ-LTA) is described for multiplexed elucidation of metalloprotein binding profiles C_LIO_LIvalidation was accomplished with the previously characterized CjNikZ; testing was accomplished with an uncharacterized homologue herein named CcSBPII C_LIO_LICcSBPII is shown to bind to multiple transition metal ions with a large range of affinities, and potentially overcome mismetallation using a simple histidine metallophore C_LI

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Diep, P., Mahadevan, R., Yakunin, A.. 2019-10-28. A microplate screen for metal-binding activity based on a nickel-binding protein from Clostridium carboxidivorans. https://doi.org/10.1101/820670

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