The Effect of the Ribosome on Nascent Chain Energy Landscapes

Abstract

Studies of protein folding in vitro have offered valuable insights into folding trajectories and kinetics; however, to date few have replicated the environment a protein encounters during translation. The first time a protein folds in vivo, it must do so co- or post-translationally, and its folding trajectory may be modulated by interactions with the ribosome exit tunnel or surface, as well as by the negative charge density associated with rRNA. Quantitatively determining how the ribosome effects folding in vivo would deepen our understanding of the requirements for efficient protein folding and offer insights into how cellular components shape the energy landscape proteins can access. Since translation involves several factors and the ribosome is a macromolecular machine, isolating and visualizing the conformations of nascent chains is challenging, making it difficult to acquire the quantitative and often atomic resolution data obtained in state-of-the-art in vitro protein folding studies. However, developments in site-specific protein labeling now make it possible to investigate complex systems by means previously only available to model proteins in optimized in vitro conditions. Here, we describe methods to establish a single-molecule Forster Resonance Energy Transfer (smFRET) signal that reports on the folded and unfolded states of a stalled nascent chain with an eye toward understanding the parameters that guide co-translational protein folding.