TY - JOUR

T1 - Quantifying metal-binding specificity of CcNikZ-II from Clostridium carboxidivorans in the presence of competing metal ions

AU - Diep, Patrick

AU - Kell, Brayden

AU - Yakunin, Alexander

AU - Hilfinger, Andreas

AU - Mahadevan, Radhakrishnan

N1 - Copyright © 2023. Published by Elsevier Inc.

PY - 2023/9/1

Y1 - 2023/9/1

N2 - Many proteins bind transition metal ions as cofactors to carry out their biological functions. Despite binding affinities for divalent transition metal ions being predominantly dictated by the Irving-Williams series for wild-type proteins, in vivo metal ion binding specificity is ensured by intracellular mechanisms that regulate free metal ion concentrations. However, a growing area of biotechnology research considers the use of metal-binding proteins in vitro to purify specific metal ions from wastewater, where specificity is dictated by the protein's metal binding affinities. A goal of metalloprotein engineering is to modulate these affinities to improve a protein's specificity towards a particular metal; however, the quantitative relationship between the affinities and the equilibrium metal-bound protein fractions depends on the underlying binding mechanisms. Here we demonstrate a high-throughput intrinsic tryptophan fluorescence quenching method to validate binding models in multi-metal solutions for CcNikZ-II, a nickel-binding protein from Clostridium carboxidivorans. Using our validated models, we quantify the relationship between binding affinity and specificity in different classes of metal-binding models for CcNikZ-II. We further illustrate the potential relevance of data-informed models to predicting engineering targets for improved specificity.

AB - Many proteins bind transition metal ions as cofactors to carry out their biological functions. Despite binding affinities for divalent transition metal ions being predominantly dictated by the Irving-Williams series for wild-type proteins, in vivo metal ion binding specificity is ensured by intracellular mechanisms that regulate free metal ion concentrations. However, a growing area of biotechnology research considers the use of metal-binding proteins in vitro to purify specific metal ions from wastewater, where specificity is dictated by the protein's metal binding affinities. A goal of metalloprotein engineering is to modulate these affinities to improve a protein's specificity towards a particular metal; however, the quantitative relationship between the affinities and the equilibrium metal-bound protein fractions depends on the underlying binding mechanisms. Here we demonstrate a high-throughput intrinsic tryptophan fluorescence quenching method to validate binding models in multi-metal solutions for CcNikZ-II, a nickel-binding protein from Clostridium carboxidivorans. Using our validated models, we quantify the relationship between binding affinity and specificity in different classes of metal-binding models for CcNikZ-II. We further illustrate the potential relevance of data-informed models to predicting engineering targets for improved specificity.

KW - Clostridium/metabolism

KW - Metals/metabolism

KW - Nickel

KW - Zinc

KW - Cobalt

KW - Metalloproteins/metabolism

KW - Protein Engineering

KW - Models, Chemical

KW - Tryptophan

KW - Fluorescence

U2 - 10.1016/j.ab.2023.115182

DO - 10.1016/j.ab.2023.115182

M3 - Article

C2 - 37355028

VL - 676

JO - Analytical biochemistry

JF - Analytical biochemistry

SN - 0003-2697

M1 - 115182

ER -