Folding of the firefly luciferase polypeptide chain with the immobilized C terminus

Abstract

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.