title: Single-molecule detection reveals knot sliding in TrmD denaturation.
abstract: An increasing number of proteins are found to contain a knot in their polypeptide chain. Although some studies have looked into the folding mechanism of knotted proteins, why and how these complex topologies form are still far from being fully answered. Moreover, no experimental information about how the knot moves during the protein-folding process is available. Herein, by combining single-molecule fluorescence resonance energy transfer (smFRET) experiments with molecular dynamics (MD) simulations, we performed a detailed study to characterize the knot in the denatured state of TrmD, a knotted tRNA (guanosine-1) methyltransferase from Escherichia coli, as a model system. We found that the knot still existed in the unfolded state of TrmD, consistent with the results for two other knotted proteins, YibK and YbeA. More interestingly, both smFRET experiments and MD simulations revealed that the knot slid towards the C-terminal during the unfolding process, which could be explained by the relatively strong interactions between the β-sheet core at the N terminal of the native knot region. The size of the knot in the unfolded state is not larger than that in the native state. In addition, the knot slid in a "downhill" mode with simultaneous chain collapse in the denatured state.
authors.affiliation: Department of Chemical Biology, College of Chemistry and Molecular Engineering, Beijing National Laboratory for Molecular Sciences, Peking University, Beijing 100871, PR China.
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.forename: Lijiang
authors.initials: L
authors.lastname: Yang
authors.forename: Pengcheng
authors.initials: P
authors.lastname: Liu
authors.forename: Yi Qin
authors.initials: YQ
authors.lastname: Gao
authors.forename: Xin Sheng
authors.initials: XS
authors.lastname: Zhao
mesh_terms: D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D008780:Methyltransferases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D011506:Proteins; D008780:Methyltransferases