Chaperones Rescue Luciferase Folding by Separating its Domains [Molecular Biophysics]

August 26th, 2014 by Scholl, Z. N., Yang, W., Marszalek, P. E.

Over the last 50 years significant progress has been made toward understanding how small single-domain proteins fold, however very little is known about folding mechanisms of medium and large multidomain proteins that predominate the proteomes of all forms of life. Large proteins frequently fold cotranslationally and/or require chaperones. Firefly (Photinus pyralis) luciferase (Luciferase) (550 residues) has been a model of a cotranslationally folding protein whose extremely slow refolding (~days) is catalyzed by chaperones. However, the mechanism by which Luciferase misfolds and how chaperones assist Luciferase refolding remains unknown. Here we combine single-molecule force-spectroscopy (AFM-SMFS) with computer simulations (SMD) to unravel the mechanism of chaperone-assisted Luciferase refolding. Our AFM and SMD results show that partially unfolded Luciferase - with the N-terminal domain remaining folded - can robustly refold without chaperones. Complete unfolding causes Luciferase to get trapped in very stable non-native configurations involving interactions between N- and C-terminal residues. However, chaperones allow the completely unfolded Luciferase to refold quickly in AFM experiments, strongly suggesting that chaperones are able to sequester non-natively contacting residues. More generally, we suggest that many chaperones, rather than actively promoting the folding, mimic the ribosomal exit tunnel and physically separate protein domains allowing them to fold in a cotranslational-like sequential process.