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Abstract

Protein folding begins co-translationally within the restricted space of the peptide exit tunnel of the ribosome. We have already shown that the N-terminal alpha-helical domain of the universally conserved N5-glutamine methyltransferase HemK compacts within the exit tunnel and rearranges into the native fold upon emerging from the ribosome. However, the exact folding pathway of the domain remained unclear. Here we analyzed the rapid kinetics of translation and folding monitored by FRET and PET using global fitting to a model for synthesis of the 112 amino acid-long HemK fragment. Our results suggest that the co-translational folding trajectory of HemK starts within the tunnel and passes through four kinetically distinct folding intermediates which may represent sequential docking of helices to a growing compact core. The kinetics of the process is defined entirely by translation. The results show how analysis of ensemble kinetics data can be used to dissect complex trajectories of rapid conformational rearrangements in multicomponent systems.