Structural proteomic analysis of folding intermediates of FLuc on the human ribosome

ABSTRACT: hRNC HDX-MS: Protein biogenesis begins on the ribosome where nascent chains are expressed vectorially from their N- to C-terminus, and start to fold as mRNA is translated. How the context of protein synthesis influences protein folding is poorly understood, especially in eukaryotes. Here we describe novel methods for the generation, purification and analysis of stable, homogeneous ribosome-bound nascent chain complexes from human cells, with the goal of defining the co-translational folding pathway of the nascent polypeptide. As a model for eukaryotic protein biogenesis, we focused on Firefly luciferase, a conformationally labile multidomain protein. Luciferase refolds slowly from denaturant, but folds rapidly in the context of translation on 80S ribosomes. Using hydrogen-deuterium exchange mass spectrometry, crosslinking-mass spectrometry and cryo-electron microscopy, we show that the human ribosome holds nascent chains in a partially unfolded state during synthesis, avoiding interdomain misfolding. This is in contrast to orthogonal bacterial RNCs, which show an overall more folded structure during synthesis, alluding to the differences observed in productive folding between the two ribosomes. Surprisingly, cotranslational folding is not strictly unidirectional. On both ribosomes, C-domain synthesis partially reverses prior folding in the N-domain. Our work facilitates a detailed mechanistic understanding of how multidomain proteins fold efficiently in human cells.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Grant Pellowe  

LAB HEAD: David Balchin

PROVIDER: PXD055280 | Pride | 2025-08-11

REPOSITORIES: Pride