Analysis of stalled ribosome-nascent chain complexes by quantitative proteomics

The cellular environment is critical for efficient protein maturation, but how proteins fold during biogenesis remains poorly understood. To understand how the cellular environment modulates folding, we studied the cotranslational chaperone-assisted folding of Escherichia coli dihydrofolate reductase (DHFR). To sample folding intermediates along the pathway of vectorial synthesis, we prepared a series of stalled ribosome:nascent chain complexes (RNCs) representing snapshots of DHFR folding in vivo. Proteomic analysis of RNCs revealed their composition and allowed us to define chaperone interactions as a function of NC length. These data set the stage for further studies aimed at resolving the conformation of the nascent polypeptide on the ribosome.