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Mechanical Unfolding and Refolding of NanoLuc via Single-Molecule Force Spectroscopy and Computer Simulations

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    Mechanical Unfolding and Refolding of NanoLuc via Single-Molecule Force Spectroscopy and Computer Simulations
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    Biomacromolecules

    Cite this: Biomacromolecules 2022, 23, 12, 5164–5178
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    https://doi.org/10.1021/acs.biomac.2c00997
    Published November 9, 2022
    Copyright © 2022 American Chemical Society
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    Abstract

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    A highly bioluminescent protein, NanoLuc (Nluc), has seen numerous applications in biological assays since its creation. We recently engineered a NanoLuc polyprotein that showed high bioluminescence but displayed a strong misfolding propensity after mechanical unfolding. Here, we present our single-molecule force spectroscopy (SMFS) studies by atomic force microscopy (AFM) and steered molecular dynamics (SMD) simulations on two new hybrid protein constructs comprised of Nluc and I91 titin domains, I91-I91-Nluc-I91-I91-I91-I91 (I912-Nluc-I914) and I91-Nluc-I91-Nluc-I91-Nluc-I91, to characterize the unfolding behavior of Nluc in detail and to further investigate its misfolding properties that we observed earlier for the I912-Nluc3-I912 construct. Our SMFS results confirm that Nluc’s unfolding proceeds similarly in all constructs; however, Nluc’s refolding differs in these constructs, and its misfolding is minimized when Nluc is monomeric or separated by I91 domains. Our simulations on monomeric Nluc, Nluc dyads, and Nluc triads pinpointed the origin of its mechanical stability and captured interesting unfolding intermediates, which we also observed experimentally.

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    Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biomac.2c00997.

    • Schematic of up-mode and down-mode operation of the AFM-based SMFS setup; WLC model fitting of recordings and ΔLc calculations; visual representation of pulling of various constructs during AFM-based SMFS experiments; partial recordings of I91-Nluc-I91-Nluc-I91-Nluc-I91; example of double peaks of I912-Nluc-I914 with all I91 domains present; iLBPs and Nluc structural comparison; AFM-based SMFS statistical analysis of the I91 titin domains from the full-length recordings of I91-Nluc-I91-Nluc-I91-Nluc-I91 and I912-Nluc-I914; small box force–extension profiles; small box force–extension profiles at 0.1 nm/ms; small box opposite pulling direction force–extension profiles; movie frames for small box with opposite pulling direction; movie frames for a large box; movie frames for a small box; movie frames for a small box at 0.25 times slower pulling speed; statistical analysis; selection process of SMFS recordings; overlapping of recordings; in silico thermal denaturation of single and triad Nluc (PDF)

    • Nluc large box simulation (AVI)

    • Nluc small box simulation (AVI)

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    Cited By

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    This article is cited by 1 publications.

    1. Dimitra Apostolidou, Pan Zhang, Devanshi Pandya, Kaden Bock, Qinglian Liu, Weitao Yang, Piotr E. Marszalek. Tandem repeats of highly bioluminescent NanoLuc are refolded noncanonically by the Hsp70 machinery. Protein Science 2024, 33 (2) https://doi.org/10.1002/pro.4895
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    Biomacromolecules

    Cite this: Biomacromolecules 2022, 23, 12, 5164–5178
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acs.biomac.2c00997
    Published November 9, 2022
    Copyright © 2022 American Chemical Society
    Request reuse permissions

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