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Reconstruction of mechanical unfolding and refolding pathways of proteins with atomic force spectroscopy and computer simulations.

Publication ,  Journal Article
Li, Q; Apostolidou, D; Marszalek, PE
Published in: Methods (San Diego, Calif.)
January 2022

Most proteins in proteomes are large, typically consist of more than one domain and are structurally complex. This often makes studying their mechanical unfolding pathways challenging. Proteins composed of tandem repeat domains are a subgroup of multi-domain proteins that, when stretched, display a saw-tooth pattern in their mechanical unfolding force extension profiles due to their repetitive structure. However, the assignment of force peaks to specific repeats undergoing mechanical unraveling is complicated because all repeats are similar and they interact with their neighbors and form a contiguous tertiary structure. Here, we describe in detail a combination of experimental and computational single-molecule force spectroscopy methods that proved useful for examining the mechanical unfolding and refolding pathways of ankyrin repeat proteins. Specifically, we explain and delineate the use of atomic force microscope-based single molecule force spectroscopy (SMFS) to record the mechanical unfolding behavior of ankyrin repeat proteins and capture their unusually strong refolding propensity that is responsible for generating impressive refolding force peaks. We also describe Coarse Grain Steered Molecular Dynamic (CG-SMD) simulations which complement the experimental observations and provide insights in understanding the unfolding and refolding of these proteins. In addition, we advocate the use of novel coiled-coils-based mechanical polypeptide probes which we developed to demonstrate the vectorial character of folding and refolding of these repeat proteins. The combination of AFM-based SMFS on native and CC-equipped proteins with CG-SMD simulations is powerful not only for ankyrin repeat polypeptides, but also for other repeat proteins and more generally to various multidomain, non-repetitive proteins with complex topologies.

Duke Scholars

Published In

Methods (San Diego, Calif.)

DOI

EISSN

1095-9130

ISSN

1046-2023

Publication Date

January 2022

Volume

197

Start / End Page

39 / 53

Related Subject Headings

  • Spectrum Analysis
  • Protein Folding
  • Peptides
  • Molecular Dynamics Simulation
  • Microscopy, Atomic Force
  • Mechanical Phenomena
  • 3101 Biochemistry and cell biology
  • 1103 Clinical Sciences
 

Citation

APA
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ICMJE
MLA
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Li, Q., Apostolidou, D., & Marszalek, P. E. (2022). Reconstruction of mechanical unfolding and refolding pathways of proteins with atomic force spectroscopy and computer simulations. Methods (San Diego, Calif.), 197, 39–53. https://doi.org/10.1016/j.ymeth.2021.05.012
Li, Qing, Dimitra Apostolidou, and Piotr E. Marszalek. “Reconstruction of mechanical unfolding and refolding pathways of proteins with atomic force spectroscopy and computer simulations.Methods (San Diego, Calif.) 197 (January 2022): 39–53. https://doi.org/10.1016/j.ymeth.2021.05.012.
Li, Qing, et al. “Reconstruction of mechanical unfolding and refolding pathways of proteins with atomic force spectroscopy and computer simulations.Methods (San Diego, Calif.), vol. 197, Jan. 2022, pp. 39–53. Epmc, doi:10.1016/j.ymeth.2021.05.012.
Journal cover image

Published In

Methods (San Diego, Calif.)

DOI

EISSN

1095-9130

ISSN

1046-2023

Publication Date

January 2022

Volume

197

Start / End Page

39 / 53

Related Subject Headings

  • Spectrum Analysis
  • Protein Folding
  • Peptides
  • Molecular Dynamics Simulation
  • Microscopy, Atomic Force
  • Mechanical Phenomena
  • 3101 Biochemistry and cell biology
  • 1103 Clinical Sciences