Molecular engineering of a fluorescent bioprobe for sensitive and selective detection of amphibole asbestos

Our plain-English summary of Molecular engineering of a fluorescent bioprobe for sensitive and selective detection of amphibole asbestos, by T. Ishida, M. Alexandrov, T. Nishimura, et al (PloS One, 2013).

The study

• Authors: T. Ishida, M. Alexandrov, T. Nishimura, R. Hirota, T. Ikeda, A. Kuroda
• Journal: PloS One
• Published: 2013;8(9):e76231
• Cited by: 6 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

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 Escherichia coli 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.

In plain English

This was a laboratory study of protein probes and mineral fibers, not a study of people. The authors set out to build a fluorescent probe that attaches to amphibole asbestos fibers so they show up under a fluorescence microscope, without attaching to wollastonite, a fibrous mineral used as an asbestos substitute. They started from H-NS, a protein from the bacterium E. coli. Pieces of H-NS were tested, and one piece, called H-NS60-90, bound amosite but not wollastonite. On its own, though, it bound only weakly. Attaching four copies of this piece to a carrier protein, streptavidin, lowered the dissociation constant from 263 nM to 1.04 nM, about 250 times lower, which means much stronger binding. Tests of shorter and altered peptides pointed to the sequence KSGTKAKR. Replacing either of its two middle lysines cut binding by 95% and 89%. The authors concluded that binding was probably electrostatic and driven by lysines, and that the probe could tell amphibole asbestos from wollastonite. They also wrote that it bound four other amphibole types, but the probe-binding tests described in the article used only amosite and wollastonite, not those four types.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: laboratory (in vitro) molecular engineering study of a fluorescent protein probe for asbestos fibers
• Who was studied: No human subjects. Mineral samples were amosite asbestos (JAWE 231) and wollastonite from Japanese reference sources, tested with proteins and peptides derived from E. coli.
• Exposure: Binding of fluorescently labeled H-NS fragments and engineered peptide tetramers to amosite asbestos (0.05 mg samples), compared with wollastonite
• Main results:
  • The H-NS fragment H-NS60-90 bound only amosite. The fragment H-NS91-137 bound both amosite and wollastonite.
  • Binding to amosite: the dissociation constant was 1.04 nM for the four-copy (tetramer) H-NS60-90 probe and 263 nM for the single-copy (monomer) H-NS60-90, about 250 times lower for the tetramer.
  • The control probes (a random peptide, free or attached to streptavidin, and streptavidin bound to biotin) showed no measurable binding to amosite.
  • The amosite-binding sequence was identified as KSGTKAKR, carried in the peptide pep3.
  • Replacing all lysines with arginines (pep8) left binding to amosite at only 6% of that of pep3.
  • Removing a single lysine reduced binding to amosite by 95% (pep11) and 89% (pep12).
  • The authors proposed that binding is probably due to electrostatic attraction between positively charged lysine side chains and negatively charged groups on the amosite surface.
• The authors' conclusion: The authors concluded that they had built a peptide probe with high affinity and specificity for amosite and for four other amphibole asbestos types, and that it could distinguish amphibole asbestos from wollastonite.
• Limitations the authors state:
  • True molar affinity cannot be calculated for binding to mineral surfaces, so the dissociation constants rest on an assumed interpretation.
  • Measuring fluorescence directly in the fiber suspension gave results that could not be reproduced, so the bound probe had to be washed off the fibers and measured separately.
  • The weak binding of the single fragments made their specificity hard to judge by fluorescence microscopy.
  • The authors did not establish the minimum linker length needed.

Topics

Medical terms in this study, explained in our Lexicon: Amphibole Asbestos, Mesothelioma, Lung Cancer, Wollastonite, Amosite, Amphibole Group.

Related studies

• Selective detection of airborne asbestos fibers using protein-based fluorescent probes — T. Ishida, M. Alexandrov, et al. (2010)
• Rapid on-site detection of airborne asbestos fibers and potentially hazardous nanomaterials using fluorescence microscopy-based biosensing — A. Kuroda, M. Alexandrov, et al. (2016)
• Detection of chrysotile asbestos by using a chrysotile-binding protein — A. Kuroda, T. Nishimura, et al. (2008)
• Development of a biosensor for detection of pleural mesothelioma cancer biomarker using surface imprinting — A. Mathur, S. Blais, et al. (2013)
• Separation and characterization of respirable amphibole fibers from Libby, Montana — J.S. Webber, D.J. Blake, et al. (2008)
• Spontaneous polymerisation on amphibole asbestos: relevance to asbestos removal — I. Fenoglio, M. Tomatis, et al. (2001)

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