RESUMEN
The refolding of thermally inactivated protein by ATP-independent trigger factor (TF) and ATP-dependent DnaKJE chaperones was comparatively analyzed. Heterodimeric (αß) bacterial luciferases of Aliivibrio fischeri, Photobacterium leiognathi, and Vibrio harveyi as well as monomeric luciferases of Vibrio harveyi and Luciola mingrelica (firefly) were used as substrates. In the presence of TF, thermally inactivated heterodimeric bacterial luciferases refold, while monomeric luciferases do not refold. These observations were made both in vivo (Escherichia coli ΔdnaKJ containing plasmids with tig gene) and in vitro (purified TF). Unlike TF, the DnaKJE chaperone system refolds both monomeric and heterodimeric luciferases with equal efficiency.
Asunto(s)
Proteínas Bacterianas/metabolismo , Luciferasas de la Bacteria/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dimerización , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Luciérnagas/enzimología , Luciferasas de la Bacteria/química , Luciferasas de la Bacteria/genética , Luciferasas de Luciérnaga/química , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Chaperonas Moleculares/metabolismo , Photobacterium/enzimología , Replegamiento Proteico , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Vibrio/enzimologíaRESUMEN
Refolding of Photinus pyralis firefly luciferase from a denatured state is a slow process; its rate and productivity depend on molecular chaperones of the Hsp70 family. In contrast, cotranslational folding of the enzyme is fast and productive in the absence of chaperones [Svetlov et al., 2006. Protein Sci. 15, 242-247]. During cotranslational folding, the C-termini of polypeptides are bound to massive particles - ribosomes. The question arises whether the immobilization of the polypeptide C-terminus on a massive particle promotes the folding. To test this experimentally, the luciferase with oligohistidine tag at its C-terminus was prepared. This allowed us to immobilize the protein C-terminal segment on chelating Sepharose beads. Here we show that both immobilized and free chains of urea-denatured enzyme refold with the same rate. At the same time, the immobilization of luciferase results in higher refolding yield due to prevention of inter-molecular aggregation. Chaperones of the Hsp70 family promote refolding of both immobilized and free luciferase polypeptides. The results presented here suggest that the high rate of cotranslational folding is not caused by the immobilization of polypeptide C-termini by itself, but is rather due to a favorable start conformation of the growing polypeptide in the peptidyl-transferase center of the ribosome and/or the strongly vectorial character of the folding from N- to C-terminus during polypeptide synthesis.
