Protein Sculpting: Probing The Interplay Between The Ribosome And Molecular Chaperones In Protein Folding In The Cell

Understanding the way in which proteins fold inside the cell is of fundamental importance to both biology and medicine. In the E. Coli cell, proteins are synthesized on the ribosome in the presence of molecular chaperones, Trigger Factor and DnaK. While both the ribosome and molecular chaperones are known to be important to protein folding in the cell, it has been nearly impossible to isolate their independent roles in the folding process since cells die at temperatures over 30C when Trigger Factor and DnaK are deleted simultaneously. In this work, we are able to access this challenging experimental condition and study the interplay between the ribosome and chaperones by synthesizing a model globin protein in a bacterial cell-free system derived from a Trigger Factor- deleted cell strain in the presence of an in-house- designed peptide inhibitor of DnaK. Surprisingly, we find that translation through the ribosome is sufficient to grant solubility to the full-length newly synthesized protein. Ongoing studies based on time-resolved fluorescence anisotropy in the frequency-domain and multidimensional nuclear magnetic resonance yield additional details on the quality of the de novo-produced protein, and on the intriguing interplay between the ribosome and chaperones in ensuring its correct folding.