Exploring the Kinetics of Protein Birth

Abstract

Fidelity and efficiency in protein folding are essential to sustain cell life. Overall however, very little is known about the way proteins are able to attain their native structure within the context of the cell. In addition to the ribosome's well-established role in peptide bond formation, recent studies suggest that ribosomes may have strong influence on the early stages of protein folding in the cell and may be crucial for the production of folded unaggregated proteins. The conformational changes that occur within a nascent protein during its release from the ribosome have yet to be elucidated. Here, we present a kinetic study on the release time-course of ribosome bound model proteins upon addition of the antibiotic puromycin. By time-resolved gel electrophoresis, we are able to discern that puromycin's hydrolysis of the ester bond linking nascent polypeptides to the 3' end of tRNA occurs quickly. Steady-state fluorescence anisotropy reveals the presence of two additional slower kinetic phases. Finally, time decay fluorescence anisotropy analysis complements the above results by providing insights into the local motions experienced by the nascent protein during different stages of the protein birth process.