The balance between protein folding and aggregation in the early stages of a protein’s life: role of the ribosome and molecular chaperones (752.6)

• Published in: The FASEB Journal
• Main Authors: Liu, Yue, Cavagnero, Silvia
• Other Authors: National Science Foundation
• Format: Article in Journal/Newspaper
• Language: English
• Published: Wiley 2014
• Subjects: Sperm whale
• Online Access: http://dx.doi.org/10.1096/fasebj.28.1_supplement.752.6

Protein biosynthesis takes place within the ribosomal machinery in the presence of the molecular chaperones trigger factor (TF) and DnaK, which are involved in the early stages of a protein’s life in bacteria. To date, the role of the ribosome, DnaK and TF in preventing co‐ and post‐translational protein aggregation is poorly understood. In this study, we used sperm whale apomyoglobin as a model protein to investigate how the ribosome, trigger factor and DnaK affect the balance between protein folding and aggregation, in the context of an E. coli cell free system. We detected both the soluble and insoluble fractions of newly synthesized apomyoglobin in a cell‐free transcription‐translation environment in the presence and absence of TF and DnaK. We took advantage of in situ labeling the N‐terminal methionine via the unnatural BODIPY‐tRNA f Met . Given that the genes encoding TF and DnaK cannot be both knocked out within the same cell strain, we developed a short peptide that can specifically inhibit DnaK activity, and added it into the cell free extract of a tig knock out strain to explore the translation in the absence of both DnaK and trigger factor. We also studied how these two chaperones and the ribosome affect incomplete nascent protein aggregation by examining the solubility of different truncated apomyoglobin constructs. Our results show that elimination of both TF and DnaK activity leads to accumulation of insoluble truncated polypeptides but does not affect the solubility of the full‐length protein. Our findings demonstrate that molecular chaperones are important for preventing any post‐translational aggregation of incomplete polypeptides but the ribosome alone suffices in promoting the successful folding of the full‐length protein. Grant Funding Source : Supported by NSF grant