Abstract
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.
Highlights
Accepted: 20 December 2020How proteins fold to their unique three-dimensional structures and how they maintain the structural conformations that are required for their biological function are central questions in biophysics [1,2,3,4,5,6,7,8]
Polyproteins are useful for low force unfolding events, which could be masked by nonspecific adhesive interactions at the beginning of the force–extension curve or confused with instrumental drift and noise
Since NanoLuc is 150x brighter than Firefly Luciferase (FLuc), these results demonstrate that our construct can be used for single brighter than FLuc, these results demonstrate that our construct can be used for single molecule studies of catalysis for concentrations as low as 16 fM
Summary
Accepted: 20 December 2020How proteins fold to their unique three-dimensional structures and how they maintain the structural conformations that are required for their biological function are central questions in biophysics [1,2,3,4,5,6,7,8]. Single-molecule force spectroscopy (SMFS) techniques are powerful tools for studying protein folding because they provide a means to directly apply forces to individual proteins under native conditions in order to measure their structural response and the internal forces that stabilize the protein [21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64] In these experiments, numerous “stretch” and “relax” cycles of force application are performed on the same protein to examine molecular elasticity within different extensions, tension regimes, and loading rates. These rearrangements that lead to high-energy conformations may reveal extremely interesting molecular properties that are not accessible to typical spectroscopic methods that usually examine biomolecules at or near their equilibrium
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