Translation: Channeling order

Proc. Natl. Acad. Sci. USA, published online 17 December 2009, doi:10.1073/pnas.0903750106

Though in vitro studies on isolated polypeptides have uncovered the chemical principles that govern protein folding, it remains unclear whether these principles apply to co-translational folding that occurs as nascent polypeptide chains (NCs) emerge from the ribosome exit channel in vivo. Cabrita et al. now report a method for isolating stalled ribosome–nascent chain (RNC) complexes from Escherichia coli and analyzing the folded states of these ribosome-bound proteins by multidimensional NMR spectroscopy. By stimulating E. coli growth to high cell densities and then inducing expression of a model protein construct in the presence of ¹⁵N-ammonium chloride and ¹³C-glucose, the authors were able to produce RNCs with the NCs labeled with NMR-active nuclei in a background of unlabeled ribosomes. The NMR spectra of the RNC consisting of full-length polypeptides were remarkably similar to those of the in vitro folded protein. However, analysis of the RNC derived from a shorter segment of the model protein, in which the domains were only partially extruded from the ribosome channel, showed more significant line broadening and fewer crosspeaks, indicative of a dynamic set of partially folded structures. In addition to providing high-resolution snapshots of co-translational folding at the ribosome, the new method offers the potential for analyzing the role that the ribosome channel and other cellular factors play in protein folding within the cell. TLS