N-Terminal Domain of Luciferase Controls Misfolding Avoidance

Abstract

The proteomes of all forms of life are predominated by large proteins. In contrast to small, one-domain proteins, little is known about folding pathways of large multidomain proteins. In this report we study Firefly (Photinus pyralis) luciferase as a model for investigating the mechanical folding and refolding of large, multidomain proteins. Using AFM-based single molecule force spectroscopy, our results indicate that Luciferase unfolds mechanically in three distinct unfolding stages. This unfolding pathway is unique as it produces additional intermediate states when the protein is extended in the presence of a single cofactor, ATP, or the pair of cofactors, D-(-)-luciferin and ATP. The modulation of the force-extension curve by ligands and denaturing chemicals, aided by computer simulations and crystallography structures, allowed us to interpret that force-extension profile as the sequential unfolding of Luciferase from the C-terminus to the N-terminus. Our results from mechanical unfolding suggest that Luciferase unfolds in a multiple-step process and that the N-terminal domain is the most stable. Interestingly, our results from single-molecule refolding experiments show that complete unfolding causes Luciferase to get trapped in non-native configurations that prevent refolding. In addition, however, these experiments show how partial unfolding of the C-terminal residues of Luciferase - with the N-terminal domain remaining folded - allow the entire protein to robustly refold, which is a novel feature of Luciferase. This feature of refolding indicates that the N-terminal domain of Luciferase may directly be involved in preventing folding into kinetic traps and provides insight on why it has evolved to fold co-translationally. These results represent an important step in understanding the folding landscapes of large proteins and have applications in drug targeting strategies and force-sensing technologies.