Nascent Proteins Interact with Key Regions of the Outer Surface of the Ribosome

Abstract

The initial stages of a protein's life in the cell are crucial for the proper balance between folding, misfolding and aggregation. While an appreciable amount of information is known about protein folding/unfolding after biosynthesis, we lack a clear understanding of how ribosome bound nascent chains (RNCs) acquire any degree of structure and dynamics. Previous studies in the Cavagnero group showed that RNCs of the intrinsically disordered protein PIR (i.e., the phosphorylated insulin receptor) and its highly negatively charged mutant PIR 1.2.3 exhibit significant spatial bias towards the ribosomal surface. We now present findings that pinpoint the presence of well-defined interactions between RNCs of PIR, PIR 1.2.3, as well as the foldable proteins bacterial flavohemoglobin apoHmpH and sperm whale apomyoglobin, with specific ribosomal proteins and the co-translationally active chaperone trigger factor (TF). RNC-ribosome contacts were probed by chemical crosslinking followed by mass spectrometry analysis, and nascent protein local and global dynamics were assessed by time-resolved fluorescence depolarization. Initial results indicate that under physiologically relevant conditions RNCs of both PIR and apoHmpH crosslink to TF in a strategic manner. In addition to interactions with TF, multiple populations of apoHmpH RNCs were observed to transiently bind to specific proteins located on the outer surface of the ribosome, both near and far from the exit tunnel. We are currently exploring the implications of these specific interactions on the balance between protein folding and aggregation. Interestingly, these interactions are strongly Mg2+ concentration-dependent. The solubility profile of apoHmpH upon release from the ribosome is not Mg2+ concentration-dependent, suggesting that the above interactions may play a yet-unidentified regulatory role in translation.