Competitive metal-binding stoichiometry between calcium and strontium by cell wall proteins of Neurospora crassa

Cell wall proteins from Neurospora crassa were isolated and evaluated to demonstrate their metal ability to bind Ca²⁺ /Sr²⁺ by loading the solubilized protein fraction on to immobilized metal affinity chromatography (IMAC) column pre-equilibrated with Ca²⁺ /Sr²⁺ . The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis IMAC eluent, revealed ∼18 proteins with a similarity in the proteome pattern of Ca²⁺ /Sr²⁺ fractions. Diethyl aminoethyl chromatography showed five proteins in common in binding to Ca²⁺ and Sr²⁺ , were subjected to N-terminal sequencing. The sequence analysis was studied for the determination of metal-binding site prediction by CHED software indicating that all five were found to have a high affinity toward Ca²⁺ . From these five, two were randomly selected and denoted as CWP-A (possess five Ca binding sites of six metal-binding sites) and CWP-B (possess six binding sites of eight metal-binding sites). They were selected for further characterization studies to determine their Ca²⁺ bound Sr²⁺ binding properties. Surprisingly, these proteins were able to bind Sr²⁺ ions (29 μmol) with equal affinity as to Ca²⁺ ions (42 μmol) by means of direct binding, and/or by displacing calcium as observed in metal-dependent proteolytic protection, fluorescence-based metal exchange assays, and molecular simulation studies. From the results, we demonstrate for the first time, that there is a stoichiometry between Ca²⁺ (an essential macro elemental metal ion) and Sr²⁺ ions (a nonessential element for which no reported metabolic activity is reported) for the metal-binding sites on cell wall proteins. This stoichiometry could be due to similar atomic dimensions and metal-protein structure stabilizing properties of Sr²⁺ compared to Ca²⁺ .