A microplate screen to estimate metal-binding affinities of metalloproteins

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A microplate screen to estimate metal-binding affinities of metalloproteins. / Diep, Patrick; Mahadevan, Radhakrishnan; Yakunin, Alexander F.
In: Analytical biochemistry, Vol. 609, 15.11.2020, p. 113836.

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Diep P, Mahadevan R, Yakunin AF. A microplate screen to estimate metal-binding affinities of metalloproteins. Analytical biochemistry. 2020 Nov 15;609:113836. Epub 2020 Aug 1. doi: 10.1016/j.ab.2020.113836

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Diep, Patrick ; Mahadevan, Radhakrishnan ; Yakunin, Alexander F. / A microplate screen to estimate metal-binding affinities of metalloproteins. In: Analytical biochemistry. 2020 ; Vol. 609. pp. 113836.

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TY - JOUR

T1 - A microplate screen to estimate metal-binding affinities of metalloproteins

AU - Diep, Patrick

AU - Mahadevan, Radhakrishnan

AU - Yakunin, Alexander F

N1 - Copyright © 2020 Elsevier Inc. All rights reserved.

PY - 2020/11/15

Y1 - 2020/11/15

N2 - 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.

AB - 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.

KW - Bacterial Proteins/chemistry

KW - Clostridium/metabolism

KW - Hydrogen-Ion Concentration

KW - Kinetics

KW - Ligands

KW - Metalloproteins/chemistry

KW - Microarray Analysis/methods

KW - Nickel/chemistry

KW - Protein Binding

KW - Recombinant Proteins/biosynthesis

KW - Spectrometry, Fluorescence

U2 - 10.1016/j.ab.2020.113836

DO - 10.1016/j.ab.2020.113836

M3 - Article

C2 - 32750358

VL - 609

SP - 113836

JO - Analytical biochemistry

JF - Analytical biochemistry

SN - 0003-2697

ER -