The long term goal of this Research Program is to understand how newly translated proteins fold in eukaryotic cells. The proposed research will focus on folding events as they occur at the ribosome during synthesis of a polypeptide and will examine the role of molecular chaperones in their folding process. The conceptual framework for understanding de novo protein folding originates from our work in the previous funding cycle, which showed that a network of chaperones named CLIPS (Chaperones Linked to Protein Synthesis) is physically and functionally linked to the translation machinery. Our working hypothesis is that the CLIPS chaperones are tasked with guiding newly synthesized polypeptides to their folded conformation. Chaperone-mediated folding pathways appear to involve the cooperation of different classes of CLIPS, including chaperones that act early in the folding process, such as the Nascent Chain Associated Complex (NAC), the Hsp70 proteins and the GIM/prefoldin complex, and the mechanistically distinct chaperone TRiC/CCT, which appears to act later in the folding process. Our general strategy to elucidate how chaperones mediate the folding of newly synthesized proteins relies on the close integration of in vitro and in vivo approaches with computational biology analyses. Our proposed experiments are aimed at obtaining functional, mechanistic and structural insights into the role of chaperones in de novo folding.