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Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc.

Publication ,  Journal Article
Ding, Y; Apostolidou, D; Marszalek, P
Published in: International journal of molecular sciences
December 2020

NanoLuc is a bioluminescent protein recently engineered for applications in molecular imaging and cellular reporter assays. Compared to other bioluminescent proteins used for these applications, like Firefly Luciferase and Renilla Luciferase, it is ~150 times brighter, more thermally stable, and smaller. Yet, no information is known with regards to its mechanical properties, which could introduce a new set of applications for this unique protein, such as a novel biomaterial or as a substrate for protein activity/refolding assays. Here, we generated a synthetic NanoLuc derivative protein that consists of three connected NanoLuc proteins flanked by two human titin I91 domains on each side and present our mechanical studies at the single molecule level by performing Single Molecule Force Spectroscopy (SMFS) measurements. Our results show each NanoLuc repeat in the derivative behaves as a single domain protein, with a single unfolding event occurring on average when approximately 72 pN is applied to the protein. Additionally, we performed cyclic measurements, where the forces applied to a single protein were cyclically raised then lowered to allow the protein the opportunity to refold: we observed the protein was able to refold to its correct structure after mechanical denaturation only 16.9% of the time, while another 26.9% of the time there was evidence of protein misfolding to a potentially non-functional conformation. These results show that NanoLuc is a mechanically moderately weak protein that is unable to robustly refold itself correctly when stretch-denatured, which makes it an attractive model for future protein folding and misfolding studies.

Duke Scholars

Published In

International journal of molecular sciences

DOI

EISSN

1422-0067

ISSN

1422-0067

Publication Date

December 2020

Volume

22

Issue

1

Start / End Page

E55

Related Subject Headings

  • Structure-Activity Relationship
  • Protein Unfolding
  • Protein Stability
  • Protein Refolding
  • Protein Folding
  • Protein Conformation
  • Microscopy, Atomic Force
  • Mechanical Phenomena
  • Luminescent Measurements
  • Luminescence
 

Citation

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ICMJE
MLA
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Ding, Y., Apostolidou, D., & Marszalek, P. (2020). Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc. International Journal of Molecular Sciences, 22(1), E55. https://doi.org/10.3390/ijms22010055
Ding, Yue, Dimitra Apostolidou, and Piotr Marszalek. “Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc.International Journal of Molecular Sciences 22, no. 1 (December 2020): E55. https://doi.org/10.3390/ijms22010055.
Ding Y, Apostolidou D, Marszalek P. Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc. International journal of molecular sciences. 2020 Dec;22(1):E55.
Ding, Yue, et al. “Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc.International Journal of Molecular Sciences, vol. 22, no. 1, Dec. 2020, p. E55. Epmc, doi:10.3390/ijms22010055.
Ding Y, Apostolidou D, Marszalek P. Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc. International journal of molecular sciences. 2020 Dec;22(1):E55.

Published In

International journal of molecular sciences

DOI

EISSN

1422-0067

ISSN

1422-0067

Publication Date

December 2020

Volume

22

Issue

1

Start / End Page

E55

Related Subject Headings

  • Structure-Activity Relationship
  • Protein Unfolding
  • Protein Stability
  • Protein Refolding
  • Protein Folding
  • Protein Conformation
  • Microscopy, Atomic Force
  • Mechanical Phenomena
  • Luminescent Measurements
  • Luminescence