The Kinetics of Nascent Protein Folding upon Release from the Ribosome

Abstract

Very little is known about the way proteins attain their native structure within the context of the living cell. In addition to the ribosome's well-established role in peptide bond formation, recent studies suggest that ribosomes play an important role in the early stages of protein folding in the cell and may be crucial for the production of folded bioactive proteins. Importantly, little is known about the impact of the mechanism of protein release from the ribosome on the attainment of a correctly folded conformation. Here, we present a kinetic study on the release time-course of fully synthesized ribosome-bound nascent proteins upon addition of the antibiotic puromycin. We focus these studies on the E. coli globin ApoHmpH. By time-resolved gel electrophoresis, we are able to follow puromycin's hydrolysis of the ester bond linking nascent polypeptides to the 3’ end of tRNA. Steady-state fluorescence anisotropy allows us to follow the escape and folding of ApoHmpH from the ribosome. Finally, time decay fluorescence anisotropy analysis in the frequency domain complements the above techniques by providing insights into the local motions experienced by the nascent protein before and after release from the ribosome. Under experimental conditions where puromycin reacts at rates comparable to the naturally occurring release factors, we show that protein release from the ribosome is rate-limited by the C-terminal ester bond cleavage, and that escape from the ribosome and completion of folding occur quickly following this step. This result shows that the ribosomal context promotes a particularly “temporally efficient” folding upon nascent protein release. An important consequence of this phenomenon is the prevention of undesirable diffusion- and concentration-dependent phenomena such as aggregation.