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

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Ding, Y; Apostolidou, D; Marszalek, P

Published Date

  • December 23, 2020

Published In

Volume / Issue

  • 22 / 1

PubMed ID

  • 33374567

Pubmed Central ID

  • PMC7801952

Electronic International Standard Serial Number (EISSN)

  • 1422-0067

International Standard Serial Number (ISSN)

  • 1422-0067

Digital Object Identifier (DOI)

  • 10.3390/ijms22010055

Language

  • eng