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Cytoskeletal Drugs Modulate Off-Target Protein Folding Landscapes Inside Cells

  • Caitlin M. Davis*
    Caitlin M. Davis
    Department of Chemistry  and  Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States
    *Email: [email protected]
  •  and 
  • Martin Gruebele*
    Martin Gruebele
    Department of Chemistry,  Department of Physics  and  Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States
    *Email: [email protected]
Cite this: Biochemistry 2020, 59, 28, 2650–2659
Publication Date (Web):June 22, 2020
https://doi.org/10.1021/acs.biochem.0c00299
Copyright © 2020 American Chemical Society

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    Abstract

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    The dynamic cytoskeletal network of microtubules and actin filaments can be disassembled by drugs. Cytoskeletal drugs work by perturbing the monomer–polymer equilibrium, thus changing the size and number of macromolecular crowders inside cells. Changes in both crowding and nonspecific surface interactions (“sticking”) following cytoskeleton disassembly can affect the protein stability, structure, and function directly or indirectly by changing the fluidity of the cytoplasm and altering the crowding and sticking of other macromolecules in the cytoplasm. The effect of cytoskeleton disassembly on protein energy landscapes inside cells has yet to be observed. Here we have measured the effect of several cytoskeletal drugs on the folding energy landscape of two FRET-labeled proteins with different in vitro sensitivities to macromolecular crowding. Phosphoglycerate kinase (PGK) was previously shown to be more sensitive to crowding, whereas variable major protein-like sequence expressed (VlsE) was previously shown to be more sensitive to sticking. The in-cell effects of drugs that depolymerize either actin filaments (cytochalasin D and latrunculin B) or microtubules (nocodazole and vinblastine) were compared. The crowding sensor protein CrH2-FRET verified that cytoskeletal drugs decrease the extent of crowding inside cells despite also reducing the overall cell volume. The decreased compactness and folding stability of PGK could be explained by the decreased extent of crowding induced by these drugs. VlsE’s opposite response to the drugs shows that depolymerization of the cytoskeleton also changes sticking in the cellular milieu. Our results demonstrate that perturbation of the monomer–polymer cytoskeletal equilibrium, for example, during natural cell migration or stresses from drug treatment, has off-target effects on the energy landscapes of proteins in the cell.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biochem.0c00299.

    • Plots of representative in vitro temperature denaturation of PGK-FRET, melt curves of PGK-FRET and VlsE-FRET collected in living U-2 OS cells, transients of PGK-FRET and VlsE-FRET, relaxation lifetimes measured at Tm versus melting temperature of VlsE-FRET, and complete tables of thermodynamic and kinetic parameters obtained in vitro and in living U-2 OS cells (PDF)

    • Fluorescence images of all cells analyzed (XLSX)

    Accession Codes

    B. burgdorferi VlsE sequence, UniProtKB 006878; Saccharomyces cerevisiae PGK sequence, UniProtKB P00560.

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    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

    Cited By

    This article is cited by 7 publications.

    1. Mark D. Ediger, Martin Gruebele, Vassiliy Lubchenko, Peter G. Wolynes. Glass Dynamics Deep in the Energy Landscape. The Journal of Physical Chemistry B 2021, 125 (32) , 9052-9068. https://doi.org/10.1021/acs.jpcb.1c01739
    2. Christopher Lambert, Katharina Schmidt, Marius Karger, Marc Stadler, Theresia E. B. Stradal, Klemens Rottner. Cytochalasans and Their Impact on Actin Filament Remodeling. Biomolecules 2023, 13 (8) , 1247. https://doi.org/10.3390/biom13081247
    3. Edward Knab, Caitlin M. Davis. Chemical interactions modulate λ 6‐85 stability in cells. Protein Science 2023, 32 (7) https://doi.org/10.1002/pro.4698
    4. Jane E. Dorweiler, Douglas R. Lyke, Nathan P. Lemoine, Samantha Guereca, Hannah E. Buchholz, Emily R. Legan, Claire M. Radtke, Anita L. Manogaran. Implications of the Actin Cytoskeleton on the Multi-Step Process of [PSI+] Prion Formation. Viruses 2022, 14 (7) , 1581. https://doi.org/10.3390/v14071581
    5. Martin Gruebele, Gary J Pielak. Dynamical spectroscopy and microscopy of proteins in cells. Current Opinion in Structural Biology 2021, 70 , 1-7. https://doi.org/10.1016/j.sbi.2021.02.001
    6. Kim Bartels, Tanya Lasitza‐Male, Hagen Hofmann, Christian Löw. Single‐Molecule FRET of Membrane Transport Proteins. ChemBioChem 2021, 22 (17) , 2657-2671. https://doi.org/10.1002/cbic.202100106
    7. Martin Gruebele. Protein folding and surface interaction phase diagrams in vitro and in cells. FEBS Letters 2021, 595 (9) , 1267-1274. https://doi.org/10.1002/1873-3468.14058

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