title: Chaperones rescue luciferase folding by separating its domains.
abstract: 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 (approximately 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 (atomic force microscopy (AFM)/single-molecule force spectroscopy) with steered molecular dynamic computer simulations to unravel the mechanism of chaperone-assisted Luciferase refolding. Our AFM and steered molecular dynamic results show that partially unfolded Luciferase, with the N-terminal domain remaining folded, can refold robustly 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.
authors.affiliation: From the Program in Computational Biology and Bioinformatics.
authors.forename: Zackary N
authors.initials: ZN
authors.lastname: Scholl
authors.affiliation: From the Program in Computational Biology and Bioinformatics, Department of Chemistry, and.
authors.forename: Weitao
authors.initials: W
authors.lastname: Yang
authors.affiliation: Department of Mechanical Engineering and Materials Science, Center for Biologically Inspired Materials and Material Systems, Duke University, Durham, North Carolina 27705 pemar@duke.edu.
authors.forename: Piotr E
authors.initials: PE
authors.lastname: Marszalek
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002457:Cell Extracts / Q000737:chemistry; D048888:Fireflies / Q000737:chemistry* / Q000201:enzymology; D049454:Firefly Luciferin / Q000737:chemistry / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018625:Microscopy, Atomic Force; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D011489:Protein Denaturation; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012156:Reticulocytes / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D002457:Cell Extracts; D019476:Insect Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; C485197:luciferase, Photinus; D049454:Firefly Luciferin; D000255:Adenosine Triphosphate; D049409:Luciferases, Firefly
keywords: Atomic Force Microscopy (AFM); Chaperone; Molecular Dynamics; Protein Folding; Single-molecule Biophysics