A miniaturized peptidyl-prolyl isomerase enzyme assay

Mirella Vivoli, Julien Renou, Arnaud Chevalier, Isobel H. Norville, Suraya Diaz, Christina Juli, Helen Atkins, Ulrike Holzgrabe, Pierre Yves Renard, Mitali Sarkar-Tyson, Nicholas J. Harmer

    Research output: Contribution to journalArticle

    2 Citations (Scopus)

    Abstract

    Prolyl-peptidyl isomerases (PPIases) are enzymes that are found in all living organisms. They form an essential part of the cellular protein folding homeostasis machinery. PPIases are associated with many important human diseases, e.g. cardiovascular disease, cancer and Alzheimer's. The development of novel PPIase inhibitors has been limited by the lack of a rapid, laboratory-based assay for these enzymes, as their substrates and products are challenging to distinguish. A well described continuous assay, coupled with the hydrolysis of a peptide by chymotrypsin is highly effective, but comparatively slow. To address this, we developed an improved version of the traditional assay using a temperature controlled plate reader. This assay allows semi-automated medium throughput assays in an academic laboratory for 84 samples per day. The assay shows lower errors, with an average Z′ of 0.72. We further developed the assay using a fluorogenic peptide-based FRET probe. This provides an extremely sensitive PPIase assay using substrate at 200 nM, which approaches single turnover conditions. The fluorescent probe achieves an excellent quenching efficiency of 98.6%, and initial experiments showed acceptable Z′ of 0.31 and 0.30 for cyclophilin A and hFKBP12 respectively. The assays provide an improved toolset for the quantitative, biochemical analysis of PPIases.

    Original languageEnglish
    Pages (from-to)59-68
    Number of pages10
    JournalAnalytical Biochemistry
    Volume536
    DOIs
    Publication statusPublished - 1 Nov 2017

    Fingerprint

    Peptidylprolyl Isomerase
    Enzyme Assays
    Assays
    Enzymes
    Isomerases
    Cyclophilin A
    Peptides
    Protein Folding
    Chymotrypsin
    Fluorescent Dyes
    Hydrolysis
    Homeostasis
    Cardiovascular Diseases
    Protein folding
    Temperature
    Substrates
    Machinery
    Quenching
    Neoplasms
    Throughput

    Cite this

    Vivoli, M., Renou, J., Chevalier, A., Norville, I. H., Diaz, S., Juli, C., ... Harmer, N. J. (2017). A miniaturized peptidyl-prolyl isomerase enzyme assay. Analytical Biochemistry, 536, 59-68. https://doi.org/10.1016/j.ab.2017.08.004
    Vivoli, Mirella ; Renou, Julien ; Chevalier, Arnaud ; Norville, Isobel H. ; Diaz, Suraya ; Juli, Christina ; Atkins, Helen ; Holzgrabe, Ulrike ; Renard, Pierre Yves ; Sarkar-Tyson, Mitali ; Harmer, Nicholas J. / A miniaturized peptidyl-prolyl isomerase enzyme assay. In: Analytical Biochemistry. 2017 ; Vol. 536. pp. 59-68.
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    abstract = "Prolyl-peptidyl isomerases (PPIases) are enzymes that are found in all living organisms. They form an essential part of the cellular protein folding homeostasis machinery. PPIases are associated with many important human diseases, e.g. cardiovascular disease, cancer and Alzheimer's. The development of novel PPIase inhibitors has been limited by the lack of a rapid, laboratory-based assay for these enzymes, as their substrates and products are challenging to distinguish. A well described continuous assay, coupled with the hydrolysis of a peptide by chymotrypsin is highly effective, but comparatively slow. To address this, we developed an improved version of the traditional assay using a temperature controlled plate reader. This assay allows semi-automated medium throughput assays in an academic laboratory for 84 samples per day. The assay shows lower errors, with an average Z′ of 0.72. We further developed the assay using a fluorogenic peptide-based FRET probe. This provides an extremely sensitive PPIase assay using substrate at 200 nM, which approaches single turnover conditions. The fluorescent probe achieves an excellent quenching efficiency of 98.6{\%}, and initial experiments showed acceptable Z′ of 0.31 and 0.30 for cyclophilin A and hFKBP12 respectively. The assays provide an improved toolset for the quantitative, biochemical analysis of PPIases.",
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    Vivoli, M, Renou, J, Chevalier, A, Norville, IH, Diaz, S, Juli, C, Atkins, H, Holzgrabe, U, Renard, PY, Sarkar-Tyson, M & Harmer, NJ 2017, 'A miniaturized peptidyl-prolyl isomerase enzyme assay' Analytical Biochemistry, vol. 536, pp. 59-68. https://doi.org/10.1016/j.ab.2017.08.004

    A miniaturized peptidyl-prolyl isomerase enzyme assay. / Vivoli, Mirella; Renou, Julien; Chevalier, Arnaud; Norville, Isobel H.; Diaz, Suraya; Juli, Christina; Atkins, Helen; Holzgrabe, Ulrike; Renard, Pierre Yves; Sarkar-Tyson, Mitali; Harmer, Nicholas J.

    In: Analytical Biochemistry, Vol. 536, 01.11.2017, p. 59-68.

    Research output: Contribution to journalArticle

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    T1 - A miniaturized peptidyl-prolyl isomerase enzyme assay

    AU - Vivoli, Mirella

    AU - Renou, Julien

    AU - Chevalier, Arnaud

    AU - Norville, Isobel H.

    AU - Diaz, Suraya

    AU - Juli, Christina

    AU - Atkins, Helen

    AU - Holzgrabe, Ulrike

    AU - Renard, Pierre Yves

    AU - Sarkar-Tyson, Mitali

    AU - Harmer, Nicholas J.

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    KW - FKBP12

    KW - Fluorescence

    KW - High throughput

    KW - Inhibitor

    KW - Peptide

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    Vivoli M, Renou J, Chevalier A, Norville IH, Diaz S, Juli C et al. A miniaturized peptidyl-prolyl isomerase enzyme assay. Analytical Biochemistry. 2017 Nov 1;536:59-68. https://doi.org/10.1016/j.ab.2017.08.004