Adsorption of the engineered peptide tetramers on amosite.

<p>Pep1-Pep7 were used to identify the binding sequence and optimal linker length. Pep8-Pep14 were used to determine the binding mechanism and the contribution of individual amino acids of the binding sequence. The percentage values indicate the adsorption of each peptide tetramer relative to that of pep3, which is referred to as the “original binding sequence”.</p

SDS–PAGE analysis of amosite-binding proteins isolated from <i>E. coli</i> lysate.

<p>SDS–PAGE analysis of amosite-binding proteins isolated from <i>E. coli</i> lysate.</p

Molecular Engineering of a Fluorescent Bioprobe for Sensitive and Selective Detection of Amphibole Asbestos

<div><p>Fluorescence microscopy-based affinity assay could enable highly sensitive and selective detection of airborne asbestos, an inorganic environmental pollutant that can cause mesothelioma and lung cancer. We have selected an <i>Escherichia coli</i> histone-like nucleoid structuring protein, H-NS, as a promising candidate for an amphibole asbestos bioprobe. H-NS has high affinity to amphibole asbestos, but also binds to an increasingly common asbestos substitute, wollastonite. To develop a highly specific Bioprobe for amphibole asbestos, we first identified a specific but low-affinity amosite-binding sequence by slicing H-NS into several fragments. Second, we constructed a streptavidin tetramer complex displaying four amosite-binding fragments, resulting in the 250-fold increase in the probe affinity as compared to the single fragment. The tetramer probe had sufficient affinity and specificity for detecting all the five types of asbestos in the amphibole group, and could be used to distinguish them from wollastonite. In order to clarify the binding mechanism and identify the amino acid residues contributing to the probe’s affinity to amosite fibers, we constructed a number of shorter and substituted peptides. We found that the probable binding mechanism is electrostatic interaction, with positively charged side chains of lysine residues being primarily responsible for the probe’s affinity to asbestos.</p> </div

Specificity of fluorescently labeled tetramers of H-NS fragments.

<p>The fibers are stained with fluorescently labeled tetramers of the indicated H-NS fragments. Each pair of phase contrast and fluorescence micrographs of amosite and wollastonite fibers shows the same field of view. Bar, 50 µm.</p

Scatchard analysis of H-NS_60-90 monomer and its streptavidin-based tetramer’s adsorption on amosite.

<p>Scatchard analysis of H-NS_60-90 monomer and its streptavidin-based tetramer’s adsorption on amosite.</p

Amosite fibers stained with fluorescently labeled monomers and streptavidin-based tetramers of H-NS_60-90.

<p>Each pair of phase contrast and fluorescence micrographs shows the same field of view. Due to lower affinity of H-NS_60-90 monomer, its adsorption on amosite at the indicated concentration is minimal. Bar, 10 µm.</p